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Early Steps in Cotranslational Translocation of Proteins across the

ER Membrane:
A Biochemical and Structural Analysis

Dissertation

zur Erlangung des akademischen Grades


d o c t o r r e r u m n a t u r a l i u m

im Promotionsfach Biologie
Spezialisierung Zellbiologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Diplom-Biochemikerin Andrea Neuhof
geboren am 7. Oktober 1971 in Nauen

Präsident der Humboldt-Universität zu Berlin: Prof. Dr. Dr. h.c. Hans Meyer

Dekan der Fakultät: Prof. Dr. Bernhard Ronacher

Gutachter: 1. Prof. Dr. Andreas Herrmann

2. Prof. Dr. Tom A. Rapoport

3. Prof. Dr. Enno Hartmann

Tag der mündlichen Prüfung: 10. Juli 2000


ZUSAMMENFASSUNG

Sekretorische Proteine und Proteine der Kompartimente des sekretorischen Transportweges


müssen die Membran des Endoplasmatischen Retikulums überqueren, um an ihren
Wirkungsort zu gelangen. In der vorliegenden Arbeit wurden frühe Schritte des
kotranslationalen Transports von Proteinen durch die ER-Membran untersucht.
Signalsequenzen leiten diese Proteine als ribosomengebundene Intermediate an die ER-
Membran. Die Ribosomen binden dort an den Sec61p-Komplex, der als Ribosomenrezeptor
wirkt und gleichzeitig den proteinleitenden Kanal in der Membran bildet. Die Assoziation von
Ribosomen mit dem Sec61p-Komplex verläuft in zwei Phasen. Die initiale Bindung ist sensitiv
gegenüber hohen Salzkonzentrationen. Die Ribosomenbindung wird salzresistent, wenn die
naszierende Kette in den Kanal inseriert und der Sec61p-Komplex die Signalsequenz
erkennt. Sowohl Ribosomen ohne naszierende Kette als auch Ribosomen, die Proteine ohne
Signalsequenzen synthetisieren, sind nur zur initialen salz-sensitiven Bindung an den
Sec61p-Komplex fähig.

Signalsequenzen interagieren im Cytosol mit SRP (engl.: Signal Recognition Particle). In


dieser Arbeit wurde gezeigt, daß Signalsequenzen außerdem von Calmodulin gebunden
werden. SRP und Calmodulin scheinen für die Interaktion mit Signalsequenzen einen
ähnlichen Mechanismus zu benutzen, der wiederum mit der Signalsequenzerkennung durch
den Sec61p-Komplex verwandt ist.

Alle Ribosomen, unabhängig davon ob und welches Protein sie translatieren, können mit dem
Sec61p-Komplex interagieren und daher um Bindungsplätze an der ER-Membran
kompetitieren. Wenn SRP an die Signalsequenz einer naszierenden Kette gebunden ist,
erhalten diese Ribosomen jedoch einen Vorteil in der Kompetition. Nur sie können
Ribosomen ohne naszierende Kette oder Ribosomen, die ein cytosolisches Protein
translatieren, vom Sec61p-Komplex verdrängen und sich selbst dann einen Translokationsort
sichern, wenn alle Bindingsplätze an der Membran besetzt sind.

In der vorliegenden Arbeit wurden dreidimensionale Strukturen von Komplexen aus Ribosom
und proteinleitendem Translokationskanal vorgestellt, die der ersten und und zweiten Phase
der Ribosomenbindung entsprechen. Überraschenderweise unterscheiden sich diese beiden
Stadien strukturell nicht. In beiden Fällen existieren definierte Verbindungen zwischen
Ribosom und Kanal, die eine Lücke von etwa 20Å zwischen dem Ribosom und der
Membranoberfläche überbrücken. Die Lücke stellt eine Verbindung zum Cytosol her, die
eventuell dazu dient, naszierende Ketten ins Cytosol zu entlassen, wenn diese nicht ins
Lumen des ER transportiert werden sollen. Weiterhin zeigen wir, daß der Kanal in nativen
Membranen größer ist als der Kanal, der nur aus gereinigtem Sec61p-Komplex besteht.
Dieser größere Kanal besitzt eine zusätzliche lumenale Domäne, die von der
Oligosaccharyltransferase oder vom TRAP-Komplex gebildet wird.

Schlagworte: Endoplasmatisches Retikulum, Proteintransport, SRP, Ribosom, Sec61p-


Komplex
SUMMARY

The first step in the secretory pathway is the translocation of proteins across the membrane
of the endoplasmic reticulum (ER). In this thesis project, early stages of cotranslational
protein translocation in mammalian cells were studied.
Proteins following the secretory pathway are targeted to the ER as ribosome-nascent chain
complexes by their N-terminal hydrophobic signal sequences. The nascent chain is
translocated across the ER membrane through a hydrophilic channel formed by the Sec61p
complex, which also functions as the ribosome receptor. The initial binding of ribosomes to
the ER membrane is salt-sensitive. After insertion of the nascent chain into the translocation
channel and signal sequence recognition by the Sec61p complex, the ribosome is bound in a
salt-resistant manner. The membrane binding of ribosomes lacking nascent chains and of
ribosomes carrying nascent chains without signal sequences is always salt-sensitive.

It is known that in the cytosol, the signal sequence binds to the signal recognition particle
(SRP). Here we show that another cytosolic factor, the small regulatory protein calmodulin,
can interact with signal sequences. Our data suggest that both SRP and calmodulin use a
similar mechanism for substrate binding and recognition. In fact, this mechanism may be
related to signal sequence recognition by the Sec61p complex.

Previously the question has been raised of how efficient targeting of ribosome-nascent chain
complexes (RNCs) carrying a signal sequence is possible when all ribosomes, regardless of
the presence or nature of a nascent chain, can bind to the Sec61p complex. We demonstrate
that all ribosomes compete for common binding sites at the ER membrane and that SRP
functions as a positive effector to give RNCs carrying a signal sequence an advantage over
other ribosomes. RNCs with a signal sequence and bound SRP can displace ribosomes
without a nascent chain and ribosomes synthesizing cytosolic proteins from the membrane
and can therefore secure a translocation site even when all ribosome binding sites at the ER
membrane are occupied.

A structural analysis by single particle cryo electron microscopy revealed that ribosome-
translocation channel complexes do not differ in the salt-sensitive or the salt-resistant stage of
ribosome binding to the ER membrane. Furthermore our data show that the ribosome is
linked to the translocation channel by a discrete number of connections. Even in the presence
of a translocating nascent chain the ribosome-membrane junction is not completely sealed
towards the cytosol. Instead, a sizable gap exists between the ribosome and the surface of
the membrane that may allow nascent polypeptide chains to enter the cytosol when their
translocation across the ER membrane is prevented. We also show that translocation
channels derived from native microsomes are larger than channels derived from purified
Sec61p complex. These larger channels contain a wider central pore and an additional
lumenal domain, which is formed by the oligosaccharyl transferase or by the TRAP complex.

Key words: Endoplasmic reticulum, Protein translocation, SRP, Ribosome, Sec61p complex
CONTENTS

1. INTRODUCTION .......................................................................................................................................1

1.1. The sorting signal for ER translocation ............................................................................................1


1.2. Cotranslational targeting of nascent polypeptides to the ER membrane .........................................2
1.3. The Sec61p complex........................................................................................................................4
1.4. The Sec61p complex forms ring-like structures ...............................................................................5
1.5. Early stages of cotranslational translocation across the ER membrane..........................................6
1.6. The signal sequence in the translocation channel ...........................................................................8
1.7. Later stages of cotranslational protein translocation across the ER membrane............................10
1.8. Biogenesis of integral membrane proteins .....................................................................................11
1.9. Regulation of ribosome-binding to the Sec61p complex................................................................12
1.10. Aims................................................................................................................................................14

2. MATERIALS AND METHODS.................................................................................................................15

2.1. Materials .........................................................................................................................................15


2.2. Preparation of microsomes ............................................................................................................16
2.3. Purification of membrane proteins and reconstitution into proteoliposomes .................................17
2.4. Preparation of ribosome-nascent chain complexes .......................................................................17
2.5. Ribosome competition ....................................................................................................................18
2.6. Insertion and translocation assays .................................................................................................18
2.7. Sample preparation ........................................................................................................................19
2.8. Photocrosslinking ...........................................................................................................................19
2.9. Preparation of bovine pancreatic cytosol and purification of calmodulin .......................................20
2.10. Preparation of ribosome-channel complexes for cryo-electron microscopy ..................................20
2.11. Identification of ribosome-associated membrane proteins.............................................................21
2.12. Electron cryo-microscopy of ribosome-channel complexes...........................................................22
2.13. Three-dimensional image processing and analysis .......................................................................22
3. RESULTS ................................................................................................................................................24

3.1. Calmodulin interacts with signal sequences ..................................................................................24


3.2. Regulation of ribosome-binding to the ER membrane ...................................................................33
3.2.1 NAC does not prevent ribosome binding to the ER membrane .....................................................33
3.2.2. RNCs carrying a signal sequence compete with nontranslating ribosomes for
membrane-binding .........................................................................................................................38
3.2.3. SRP-independent targeting in the reticulocyte lysate system ........................................................42
3.2.4. SRP-independent targeting to reconstituted proteoliposomes.......................................................44
3.2.5. Binding of the signal sequence to the Sec61p complex is necessary for gaining
a competitive advantage.................................................................................................................46
3.2.6. SRP binding gives a competitive advantage to ribosomes synthesizing integral
membrane proteins with a signal anchor .......................................................................................48
3.2.7. Membrane binding of ribosomes synthesizing cytosolic proteins ..................................................50
3.2.8. Ribosome binding during integration of multispanning membrane proteins ..................................52
3.3. Structural analysis of ribosome binding to the ER membrane .......................................................54
3.3.1. Preparation of ribosome-translocation channel complexes ...........................................................54
3.3.2. Structures of ribosomes bound to purified Sec61p complex..........................................................57
3.3.3. Ribosomes bound to native translocation channels.......................................................................59
3.3.4. Ribosome-channel complexes with a mixed population of nascent chains ...................................62
3.3.5. The ribosome-channel junction ......................................................................................................63
3.3.6. Comparison of purified Sec61p channel and native translocation channel ...................................67
3.3.7. The nature of the lumenal domain..................................................................................................69

4. DISKUSSION...........................................................................................................................................71

4.1. Interaction of calmodulin with signal sequences ............................................................................71


4.1.1. Substrate binding by calmodulin ....................................................................................................71
4.1.2. Protein translocation and calmodulin .............................................................................................73
4.2. Regulation of ribosome binding .....................................................................................................74
4.2.1. SRP-independent targeting in the presence of cytosol ..................................................................75
4.2.2. How does an SRP-advantage work? .............................................................................................77
4.2.3. Other aspects of unspecific ribosome binding to the ER ...............................................................79
4.2.4. Consequences for integration of multispanning membrane proteins.............................................80
4.3. 3D structures of ribosome-translocation channel complexes ........................................................81
4.3.1. The ribosome-membrane junction..................................................................................................81
4.3.2. Shape and size of the translocation channel .................................................................................85
4.3.3. A lumenal domain associated with the native Sec61p channel .....................................................86

5. REFERENCES ........................................................................................................................................89

6. APPENDIX...............................................................................................................................................98

Abbreviations ..................................................................................................................................98
Erklärung ........................................................................................................................................99
Lebenslauf ....................................................................................................................................100
Veröffentlichungen........................................................................................................................101
Danksagung .................................................................................................................................102
Eigenanteil an der vorgelegten Arbeit ..........................................................................................103
Introduction

1. INTRODUCTION

Eukaryotic cells contain membrane-enclosed compartments, such as mitochondria, nuclei


and lysosomes. Each of these organelles is equipped with a unique set of proteins to fulfill
specific tasks within the cell. Therefore, sorting of proteins to their correct location is a critical
step for establishing and maintaining the identity of organelles.

Proteins destined to be secreted from the cell or for residence in the plasma membrane, the
lysosomes, the Golgi apparatus or the endoplasmic reticulum (ER) follow the secretory
pathway to their destination. The first step of the secretory pathway is targeting of the nascent
protein to the membrane of the endoplasmic reticulum and subsequent translocation across
the membrane into the ER lumen or, in case of membrane proteins, the integration into the
phospholipid bilayer (Palade, 1975). During later stages of the secretory pathway transport
vesicles shuttle proteins from the ER to the plasma membrane and lysosomes via the Golgi
apparatus.

Protein translocation across the ER membrane (for review see Rapoport et al., 1996a) can
occur while the protein is being synthesized by the ribosome (cotranslational translocation) or
after translation has been completed (posttranslational translocation). In mammalian cells
most proteins are translocated cotranslationally. In the yeast Saccharomyces cerevisiae,
however, both the co- and the posttranslational pathway are used.

1.1. The Sorting Signal for ER Translocation

The signal for ER-targeting is an N-terminal hydrophobic peptide of approximately 15-30


residues (Blobel and Dobberstein, 1975; for review see Gierasch, 1989; Martoglio and
Dobberstein, 1998). The primary structure of these signal sequences is not conserved,
instead they share characteristic features (von Heijne, 1985). A hydrophobic core of about 8-
15 residues is preceded by a short polar region (N-domain) which may contain charged
amino acids.

1
Introduction

The region following the hydrophobic stretch generally contains small polar residues. The
hydrophobic core is thought to form an α-helix (von Heijne, 1985). It has also been shown
that isolated signal peptides can adopt an α-helical conformation in nonpolar environments
(Gierasch, 1989; McKnight et al., 1989).

1.2. Cotranslational Targeting of Nascent Polypeptides to the ER Membrane

In cotranslational translocation, targeting to the membrane of the endoplasmic reticulum


begins when the signal sequence of the nascent polypeptide chain emerges from the
ribosome (for review see Walter and Johnson, 1994). A cytosolic factor, the signal recognition
particle (SRP) has been shown to bind to signal sequences (Walter et al., 1981). SRP
consists of the 7SL RNA and six polypeptides (Walter and Blobel, 1980; 1982). The 54kDa
subunit of SRP (SRP54) interacts with signal sequences (Krieg et al., 1986; Kurzchalia et al.,
1986). SRP54 comprises a GTPase domain (Bernstein et al., 1989; Römisch et al, 1989) and
a methionine-rich M-domain that has been shown to contain the signal sequence binding site
(Zopf et al., 1990; High and Dobberstein, 1991; Lütcke et al., 1992). It is thought that the
highly flexible side chains of methionines line the walls of a hydrophobic pocket, forming a
“methionine- bristle” that would allow recognition of a wide variety of signal peptides
(Bernstein et al., 1989). A structural analysis of a bacterial homologue of SRP54 confirmed
the existence of a hydrophobic groove in the M-domain (Keenan et al., 1998).

Ribosomes carrying a nascent polypeptide with bound SRP are targeted to the endoplasmic
reticulum by two affinities: The ribosome interacts with its membrane receptor, the Sec61p
complex (Görlich et al., 1992a; Kalies et al., 1994; Jungnickel and Rapoport, 1995), and SRP
interacts with the SRP receptor (SR; Gilmore et al., 1982a; 1982b; Meyer et al., 1982). After
binding of SRP to SR, the signal sequence is released from SRP54 (Connolly and Gilmore,
1989; Rapiejko and Gilmore, 1997). It is now free to contact the translocation site at the ER
membrane (translocon) and subsequently protein translocation is initiated.
The SRP receptor has two subunits, SRα and SRβ. Like SRP54, they both contain GTPase
domains (Connolly and Gilmore, 1989; Miller et al., 1995). In fact, an analysis

2
Introduction

of the crystal structure of Ffh, the bacterial homologues of SRP54, and of FtsY, the
homologue of SRα, suggests that their GTPase domains are related and constitute their own
subfamily of GTPases (Freymann et al., 1997; Montoya et al., 1997).

A
B
signal sequence translocon SRP receptor

SRP C
F

E
D

Figure 1: SRP-mediated targeting. A: When a signal sequence emerges from the ribsosome, it is bound by the
nucleotide-free form of the 54kDa subunit of signal recognition particle (SRP54). B: The ribosome-nascent chain
complex with bound SRP is then targeted to the ER membrane by two interactions: SRP binds to the SRP receptor
(SR) and the ribosome contacts the Sec61p complex. C: The affinity of SRP54 for GTP is increased upon binding to
SR. D: GTP binding to SRP54 results in the release of the signal sequence which can now interact with the α subunit
of the Sec61p complex. Subsequently, translocation begins. SRP-GTP remains bound to SR. In the GTP-bound
state, SRP54 and SRα reciprocally stimulate their GTPase activity. E: After GTP hydrolysis, SRP dissociates from SR
and releases GDP. F: SRP54 remains in a nucleotide-free form until SRP engages in the next round of targeting.

The GTP cycles of SRP54 and SRα regulate targeting of RNCs to the ER membrane (Figure
1; see Rapiejko and Gilmore, 1997): According to a current model nucleotide-free SRP54
binds to a signal sequence (Rapiejko and Gilmore, 1997). The complex is thought to remain
in a state of low affinity for GTP. Interaction of SRP with the ribosome might increase the
affinity of SRP for GTP (Bacher et al., 1996). A further increase occurs upon targeting of the
RNC-SRP to the ER membrane and interaction of SRP with its receptor (Miller et al., 1993;
Rapiejko and Gilmore, 1997). It has been proposed that GTP-binding to SRP54 causes a
conformational change that results in the release of the signal sequence, allowing
translocation to begin (Connolly and Gilmore, 1989). SRP-GTP remains associated with SR
and would dissociate only after GTP hydrolysis (Connolly et al., 1991). It has been shown that
SRP54 and SRα act as

3
Introduction

GTPase-activating proteins for each other while they are in their GTP-bound form (Powers
and Walter, 1995). After GTP hydrolysis SRP54 releases GDP and remains in a nucleotide-
free form until the next round of targeting (Connolly et al., 1991; Rapiejko and Gilmore, 1997).
The release of GDP without a nucleotide exchange factor and the stable nucleotide-free
stage of SRP54 (Freymann et al., 1997; Freymann et al., 1999) are an unusual feature of this
GTPase.

1.3. The Sec61p Complex

Both the release of the signal sequence from SRP54 and the binding of the translating
ribosome to the ER membrane are prerequisites for the nascent chain to engage in
translocation. But how does a hydrophilic polypeptide cross a hydrophobic membrane? A
protein-conducting channel in the membrane of the endoplasmic reticulum had been
proposed (Blobel and Dobberstein, 1975; Gilmore and Blobel, 1985) and electrophysiology
studies have provided evidence for its existence (Simon and Blobel, 1991). Furthermore,
fluorescence quenching data suggested that the nascent chain is in an aqueous environment
while it is crossing the membrane (Crowley et al., 1993). The heterotrimeric Sec61p complex
has been identified as a central component of the translocation machinery. In mammals, the
Sec61p complex contains an α subunit with 10 membrane-spanning domains, and β and γ
subunits, each of which spans the membrane once (Figure 2; Hartmann et al., 1994). Initially,
crosslinking experiments provided evidence that the Sec61p complex forms the actual
channel (Mothes et al., 1994; for review see Martoglio and Dobberstein, 1996). The Sec61p
complex has been found to be tightly associated with membrane-bound ribosomes and it also
functions as the ribosome receptor (Görlich et al., 1992a; Kalies et al., 1994). Using a
photocrosslinking approach it has been shown that the α subunit of the Sec61p complex is in
proximity to nascent polypeptide chains throughout their transfer across the membrane
(Mothes et al., 1994).

4
Introduction

N H2 Sec61α Sec61β Sec61γ


C OOH
N H2 N H2
c y t o so l

l ume n C OOH C OOH

Figure 2: The membrane topology of the mammalian Sec61p complex. Sec61α is an integral membrane protein with
10 membrane spanning domains. It is highly homologous among different species. The conserved regions are shown
in red. Sec61β and Sec61γ are tail-anchored membrane proteins with their only membrane spanning domain located
at the C-terminus.

Additional evidence that the Sec61p complex forms the translocation channel came from
experiments utilizing reconstituted proteoliposomes. Purified membrane proteins and pure
lipids can be used to generate proteoliposomes of a defined composition. The Sec61p
complex and SRP receptor have been shown to be necessary and sufficient for translocation
of the secretory proteins preprolactin, Kar2p and pre-growth hormone (Görlich et al., 1992a;
Voigt et al., 1996). Since the function of SR is SRP binding and since SR only contains one
membrane-spanning domain, it has been concluded that the Sec61p complex forms the
protein conducting channel and that it acts as the ribosome receptor.

The Sec61p complex is evolutionary well conserved from bacteria to man (Hartmann et al.,
1994; for review see Matlack et al., 1998). In the budding yeast S. cerevisiae, Sec61p, a
homologue of Sec61α, was found in a genetic screen (Deshaies and Schekman, 1987). It
forms the Sec61p complex together with Sbh1p and Sss1p, the homologues of mammalian
Sec61β and γ. In E. coli, SecY and SecE are homologues of Sec61α and Sec61γ,
respectively. Together with SecG, they form the SecYEG complex, the major component of
the translocation channel in the bacterial inner membrane (Hanada et al., 1994; for review
see Ito, 1995).

1.4. The Sec61p Complex Forms Ring-Like Structures

Further evidence that the Sec61p complex forms the protein conducting channel in the ER
membrane comes from electron microscopy studies. The purified Sec61p complex from
mammals and S. cerevisiae and the purified SecYEG complex all assemble into ring-like
structures in detergent solution (Hanein et al., 1996; Meyer et

5
Introduction

al., 1999; Manting et al., 2000). The size of these rings suggest that each contains three or
four copies of the Sec61p or SecYEG complex, in good agreement with the finding that in
mammals membrane-bound ribosomes are associated with 3 to 4 copies of the Sec61p
complex (Hanein et al., 1996).
Channel-like structures have also been detected in native ER membranes examined by
freeze-fracture electron microscopy (Hanein et al., 1996; Meyer et al., 1999). Yet, when
purified mammalian Sec61p complex was reconstituted into proteoliposomes, no rings were
seen. However, the ring-like structures reappeared when the membranes were incubated
with ribosomes before electron microscopy. This was confirmed with proteoliposomes
containing the yeast Sec61p complex. Again, ring-like structures were seen after addition of
ribosomes to the vesicles. Interestingly, the same structures appeared after coreconstitution
of the Sec62/63p complex, a tetrameric complex of membrane proteins that interacts with the
Sec61p complex to facilitate posttranslational translocation of proteins (Deshaies et al.,
1991). This suggests that oligomerization of the Sec61p complex may be induced by the
interacting partner(s) in either co- or posttranslational protein transport.

Recently, a structure of yeast nontranslating ribosomes bound to S. cerevisiae Sec61p


complex in detergent solution was determined by single particle cryo electron microscopy
(Beckmann et al., 1997). This 3D map shows a channel very similar to the ones described
above. The ribosome is separated from the Sec61 channel by a sizable gap, bridged only by
a single connection. One goal of this thesis was to determine the structure of a ribosome-
nascent chain-Sec61p complex to ascertain whether the ribosome-channel junction becomes
more intimate when a nascent chain is inserted into the translocation channel.

1.5. Early Stages of Cotranslational Translocation Across the ER Membrane

Ribosome-nascent chain complexes are targeted to the endoplasmic reticulum by the SRP
pathway and by the affinity of the ribosome for the Sec61p complex. Using early translocation
intermediates two distinct stages of ribosome-membrane interaction have been characterized.

6
Introduction

First the ribosome-nascent chain complex (RNC) is bound only loosely to the Sec61p
complex (Figure 3A; Jungnickel and Rapoport, 1995). This initial ribosome-Sec61p interaction
is salt-sensitive (see also Wolin and Walter, 1993) and the nascent chain is accessible to
cytosolic proteases. However, even at this early stage the signal sequence contacts the α
subunit of the Sec61p complex, as shown by photocrosslinking experiments. Upon elongation
of the nascent chain, at a length of about 70 residues for the secretory protein preprolactin, a
transition to tight binding of the RNC to the channel takes place (Figure 3B; Jungnickel and
Rapoport, 1995).

A loose B tight

signal sequence

Sec61p

Figure 3: Loose and tight stages of ribosome binding to the Sec61p complex. A: The initial interaction between a
ribosome-nascent chain complex carrying a signal sequence and the Sec61p complex is sensitive to high salt
concentrations. The nascent chain remains sensitive to protease digestion. B: Upon elongation of the nascent chain,
the signal sequence inserts into the translocation channel and is recognized by the Sec61p complex. The interaction
between ribosome and Sec61p channel is now resistant to high salt concentration and the nascent chain becomes
protected from protease digestion.

The interaction between ribosome and Sec61p complex becomes resistant to high salt
concentrations (Wolin and Walter, 1993; Connolly and Gilmore, 1986). Furthermore, the
nascent chain is protected against added proteases. Fluorescent probes incorporated into the
nascent chain cannot be quenched by reagents added from the cytosolic side of the ER but
they become accessible to quenching reagents that gained access to the lumen of the ER
(Crowley et al., 1993; 1994). This suggests that after the transition from loose to tight
ribosome binding to the ER membrane one continuous channel exists from the peptidyl-
transferase center in the ribosome to the lumen of the ER.
Only ribosome-nascent chain complexes containing a functional signal sequence undergo the
transformation from a loose to a tight state of ribosome-membrane interaction (Jungnickel
and Rapoport, 1995). Therefore this transition comprises the following events:
- Recognition of the signal sequence by the translocation channel
- A shift to high salt resistant binding of the ribosome to the Sec61p complex
- Closing of the ribosome-membrane junction to the cytosol
- Opening of the channel towards the lumen of the endoplasmic reticulum

7
Introduction

All events are triggered by binding of the signal sequence to the Sec61p complex. This
occurs when the nascent chain has reached a length such that the signal sequence can
interact productively with the Sec61 channel. It has been suggested that the nascent chain
inserts into the translocation channel as a loop with its N-terminus located in the cytosol (see
Figure 3B; Shaw et al., 1988; Mothes et al., 1994). The critical length of the nascent chain for
signal sequence recognition by the Sec61p complex varies slightly for different substrates
(Mothes et al., 1998), possibly reflecting differences in the length of the signal peptide and of
the hydrophobic core.

So far, the molecular mechanisms are unknown by which the transition from loose to tight
ribosome binding occurs. The insertion of a nascent chain into the translocation channel
could cause a conformational change that increases the strength of the ribosome-channel
interaction, or the chain could merely provide an additional, stabilizing link. The 3D map of the
yeast nontranslating ribosome bound to the yeast Sec61p complex (Beckmann et al., 1997)
most likely visualizes the ribosome-channel junction in the loose binding stage. In this
structure, a large gap is seen between the ribosome and the Sec61p complex. However, the
biochemical data (Crowley et al., 1994) might suggest that in the tight binding state the
ribosome-membrane interaction would become much more intimate and that a continuous
sealing exist around the ribosome-membrane junction. Therefore, one of the most exciting
questions asked in this thesis is whether structural differences can be detected in 3D maps of
ribosome-channel complexes with and without a nascent chain.

1.6. The Signal Sequence in the Translocation Channel

Using site-specific photocrosslinking, the environment of signal sequences in translocation


intermediates after signal sequence recognition by the Sec61p complex has been mapped
(Plath et al., 1998). The signal sequence contacts Sec61α and seems to be precisely
oriented with respect to the Sec61p complex. The crosslinking pattern suggests that in the
translocon the signal sequence adopts an α-helical structure. The signal sequence has been
shown to be in close proximity to proteinaceous components of the translocation channel as
well as to lipid molecules,

8
Introduction

indicating that it is located at the interface of protein channel and lipid phase (Mothes et al.,
1998). A similar study of signal sequence recognition in posttranslational translocation in
S.cerevisiae supported these results (Plath et al., 1998). In addition, these data revealed that
the membrane spanning domains 2 and 7 of the yeast Sec61α homologue face the binding
site and sandwich the α helix formed by the signal sequence.

In the mammalian cell, another component of the translocon, the translocating chain
associated membrane (TRAM) protein, interacts with the signal sequence (Görlich et al.,
1992b; High et al., 1993; Plath et al., 1998). It seems that TRAM contacts one side of the
signal sequence when it is inserted into the translocation channel as an α-helix. Interestingly,
the same side is interacting with Sec62p in the posttranslational translocation pathway in
yeast (Plath et al., 1998).
Experiments employing reconstituted proteoliposomes containing purified membrane proteins
have shown that TRAM is essential for translocation of most secretory and membrane
proteins (Görlich et al., 1992b; Görlich and Rapoport, 1993; Voigt et al., 1996). However, the
function of TRAM in protein translocation across the membrane of the ER remains elusive.
TRAM dependence or independence is conferred by the signal sequence. Proteins containing
a long and very polar N-region and a long hydrophobic core rich in leucine and valine
residues are generally TRAM-independent (Voigt et al., 1996).

Another component of the translocon that has been found in proximity to the signal sequence
(Wiedmann et al., 1987) and to mature regions of the nascent chain during translocation
(Krieg et al., 1989; Wiedmann et al., 1989) is the heterotetrameric translocon associated
protein (TRAP) complex. TRAP has also been shown to be tightly associated to the
membrane bound ribosome (Görlich et al., 1992a). Although the TRAP complex seems to be
an integral part of the translocon it is not essential for translocation of all proteins tested so far
(Migliaccio et al., 1992; Görlich and Rapoport, 1993). For the secretory protein preprolactin it
has been demonstrated that TRAP does not even stimulate translocation (Görlich and
Rapoport, 1993).

9
Introduction

1.7. Later Stages of Cotranslational Protein Translocation Across the ER


Membrane

As the nascent chain is elongated further, the signal sequence is cleaved off by the signal
peptidase complex at a recognition site following the hydrophobic core (Evans et al., 1986).
After cleavage, the signal peptide remains in the lipid bilayer where it is subsequently
processed by a signal peptide peptidase. Fragments of the signal sequence are then
released into the cytosol (Lyko et al., 1995).
The oligosaccharyl transferase complex transfers carbohydrate chains from
dolicholphosphate onto glycosylation sites of the nascent chain (Kelleher et al., 1992). Other
ER resident proteins presumably interacting with newly translocated polypeptides are protein
disulfide isomerase (PDI), which facilitates the formation of disulfide bonds, prolylpeptidyl
cis/trans isomerase, which promotes prolyl cis/trans isomerization, and chaperones in the ER
lumen, assisting in folding of translocated polypeptides.

In contrast to early stages of translocation, which are well characterized, little is known about
how translocation is terminated. It has been suggested that release of the nascent chain and
dissociation of the ribosome from the translocation site are necessary for closing of the
translocation channel (Simon and Blobel, 1991). Yet, ring-like structures formed by the
Sec61p complex were observed in microsomes that had been treated with puromycin under
high salt conditions to release nascent chains and remove membrane bound ribosomes
(PKRM; Hanein et al., 1996). Thus, Sec61p channels may remain assembled in a closed
conformation after termination of translocation and release of the ribosome. Alternatively,
puromycin-induced termination may not result in disassembly of the channel that may occur
under physiological conditions. Strikingly, solubilization of PKRM with digitonin yields intact
Sec61p complexes that can promote protease-resistant insertion of short translation
intermediates (Mothes et al., 1998). Additional membrane proteins have been shown to be
tightly associated with membrane bound ribosomes after detergent solubilization of rough
microsomes (Görlich et al., 1992a) suggesting that they may be part of a larger translocation
complex that assembles for each round of translocation and disassembles after translocation
has been terminated.

10
Introduction

1.8. Biogenesis of Integral Membrane Proteins

Membrane proteins destined to reside in the plasma membrane, lysosomes, the Golgi
network or the ER also follow the secretory pathway (for review see Hegde and Lingappa,
1997; Matlack et al., 1998) and use the same translocation machinery as soluble proteins
(Görlich and Rapoport, 1993; Oliver et al., 1995). First, the precursors are targeted to the ER
membrane as ribosome-nascent chain complexes. For targeting, membrane proteins may
use a cleavable signal peptide or a noncleavable signal anchor. Signal anchors are generally
longer and more hydrophobic than signal peptides (for review see von Heijne and Manoil,
1990). Furthermore, they lack the recognition site for the signal peptidase. SRP interacts with
both signal anchors and signal sequences of integral membrane proteins. After targeting,
nascent membrane proteins are translocated across the ER membrane and their one or more
membrane spanning domains are integrated into the phospholipid bilayer in the correct
orientation.

It is not clear yet at which point a membrane anchor is transferred from the proteinaceous
channel to the lipid phase. It has been suggested that membrane spanning domains either
remain in a proteinaceous environment until termination of translation and release of the
nascent chain from the ribosome (Borel and Simon, 1996; Do et al., 1996) or that membrane
anchors contact phospholipids in an early phase of translocation, suggesting that even during
translocation the Sec61p channel opens laterally towards the lipid phase (Martoglio et al.,
1995; Mothes et al., 1997).
Another open question is how the translocation channel is gated during biogenesis of an
integral membrane protein. According to one study, the channel may be closed towards the
ER lumen and opened towards the cytosol while the membrane anchor is still buried within
the translating ribosome (Liao et al., 1997), implying that the ribosome can recognize a
membrane anchor and induce a conformational change of the translocation channel. This
would be very different from the gating of the channel that is induced by binding of the signal
sequence to the Sec61p complex (Crowley et al., 1994; Jungnickel and Rapoport, 1995).

11
Introduction

1.9. Regulation of Ribosome-Binding to the Sec61p complex

It has been shown that nontranslating ribosomes have an intrinsic affinity for microsomal
membranes (Borgese et al., 1974; Kalies et al., 1994). At the ER membrane, the Sec61p
complex functions as the ribosome receptor (Görlich et al., 1992; Kalies et al., 1994). It has
also been shown that ribosome-nascent chain complexes (RNCs) can be targeted to the ER
membrane by the interaction of ribosomes and translocation sites alone (Lauring et al.,
1995b; Jungnickel and Rapoport, 1995). If all ribosomes, independent of the nature or
presence of a nascent chain, can bind to translocation sites at the ER membrane, how does
efficient targeting occur for RNCs carrying a signal sequence?
Recently, a model invoking a cytosolic inhibitor of ribosome binding to the Sec61p complex
has been proposed (Wiedmann et al., 1994). This model suggests that the inhibitor, nascent
polypeptide-associated complex (NAC) binds to nascent chains emerging from the ribosome
and to nontranslating ribosomes. The presence of NAC on RNCs (Lauring et al., 1995a) or on
nontranslating ribosomes (Möller et al., 1998) is thought to prevent binding to SRP and to the
ER membrane. In the case of nascent chains with a signal sequence, SRP would be able to
compete off NAC by specific binding to the signal sequence (Lauring et al., 1995b), allowing
the ribosome-nascent chain complex to be targeted to the membrane.
According to this model, only ribosomes carrying a nascent chain with a signal sequence and
bound SRP are targeted to the ER membrane. Signal sequence recognition by the Sec61p
complex during subsequent stages of translocation would merely be a double check.

The role of NAC in regulation of ribosome binding to the ER is somewhat controversial. First
of all, a substantial population of ribosomes is bound to rough microsomes in a high salt
sensitive manner (about 45%, Kalies et al., 1994), indicating that they are not engaged in
translocation. It is unlikely that such a large fraction presents ribosome-nascent chain
complexes in early, salt-sensitive, stages of translocation. Furthermore, one study affirmed
the specificity of SRP for signal sequence-bearing RNCs (see Walter et al., 1981) in the
presence and absence of NAC (Powers and Walter, 1996). Besides, all data supporting the
NAC model were derived

12
Introduction

from in vitro experiments using RNCs that had been washed with a high salt buffer to remove
NAC and SRP. It has been shown that these high salt washed RNCs can bind to microsomes
in a manner that is independent of SRP and the presence of a signal sequence. Readdition of
NAC restored SRP and signal sequence dependent binding. So far, the role of NAC in
regulation of ribosome binding to the ER membrane has not been studied under more
physiological conditions.
Another argument against the proposed role of NAC in regulation of ribosome targeting
comes from experiments in S. cerevisiae. The deletion of NAC homologues in yeast does not
result in a growth defect or in a secretion phenotype (Reimann et al., 1999). Instead there are
reports that in S. cerevisiae NAC is involved in protein targeting to mitochondria (George et
al, 1998; Fünfschilling and Rospert, 1999) and that NAC homologues are functioning as
transcription factors for GAL4 (Shi et al., 1995). The function of NAC as a transcriptional co-
activator for GAL4/VP-16 was confirmed in mammalian cell lines (Yotov et al., 1998). It was
also reported that the α subunit of NAC potentiates c-Jun mediated transcription (Moreau et
al., 1998).

One aim of this thesis was to re-evaluate the function of NAC by studying SRP-independent
targeting of signal sequence-bearing RNCs in a full translation system. We also wished to
determine which role SRP binding to the signal peptide plays and how signal sequence
recognition by the Sec61p complex is important in achieving specificity of targeting.

13
Introduction

1.10. Aims

The thesis project presented here explores different aspects of initial steps in cotranslational
protein translocation.

1. The N-terminal hydrophobic signal sequence is known to bind to the cytosolic


signal recognition particle (SRP). Yet, since in the cell there is only one SRP for every 10-100
ribosomes (Walter and Johnson, 1994; Ogg and Walter, 1995) it is conceivable that
hydrophobic signal peptides can interact with cytosolic factors other than SRP; either during
SRP-independent targeting of RNC to the ER membrane or to prevent aggregation in a
hydrophilic milieu before SRP binding. Using a photocrosslinking approach we wanted to
probe the cytosolic environment of the signal sequence and identify possible binding partners.

2. All ribosomes, independent of the presence or nature of a nascent chain, can


interact with the Sec61p complex. Thus, we wished to address the question how under these
conditions specificity in targeting is achieved. We were interested in assessing the role of
cytosolic factors in regulation of RNC-binding to the Sec61p complex. In addition, we wanted
to compare binding of ribosomes with or without various nascent chains in competition
assays.

3. Two stages of ribosome binding to the Sec61p complex are known: an initial loose
binding and a tighter binding after insertion of a nascent chain into the translocation channel
and subsequent signal sequence recognition. Using single particle cryo electron microscopy,
we carried out a structural comparison of ribosome-channel complexes with and without a
nascent chain, representing the tight and loose binding state, respectively. We also wanted to
compare the structures of the native translocation channel and of the channel formed by the
isolated Sec61p complex since it is known that other membrane proteins in addition to the
Sec61p complex are part of the translocon.

14
Materials and Methods

2. MATERIALS AND METHODS

2.1. Materials

-Chemicals and enzymes were purchased from Sigma-Aldrich, Fisher Scientific, New
England Biolabs, Inc., Biorad Laboratories, Promega Corp., Pierce Chemical Company,
Amersham Pharmacia Biotech, EM Science, Roche Molecular Biochemicals, Calbiochem
Corp. and Avanti Polar Lipids Inc.

-Antibodies were raised in rabbits against peptides of Sec61α (CKEQSEVGSMGALLF),


Sec61β (PGPTPSGTNC), TRAPα (CLPRKRAQKRSVGSDE), TRAM (CADSPRNRKEKSS)
by BabCo and Cocalico Corp. Antibodies directed against the α subunit of NAC were a gift
from Dr. M. Wiedmann, antibodies against the ribosomal protein S26 were a gift from Dr. J.
Stahl.

-Truncated mRNAs coding for nascent chains were generated by in vitro transcription as
described before:
ppl86, ppl59, ppl86∆13-15 Jungnickel and Rapoport, 1995
ppl132 Mothes et al., 1994
ppl169 Görlich et al., 1992
ppαFt86amb8 to 16, ppαFt86K5 Plath et al., 1998
lep57, lep68, lep70, lepcyt, lepXa Mothes et al., 1997
Invertase Voigt et al., 1996
ffl77 Wiedmann et al., 1994

-Photocrosslinking reagents were prepared as described in Görlich et al., 1991 (TDBA-lysyl


tRNA) and Martoglio et al., 1995 (TmD-Phe suppressor tRNA).

-Purified NAC was a gift from Dr. M. Wiedmann. Calmodulin purified from bovine brain was
purchased from Calbiochem Corp. SRP was purified as described in Walter and Blobel,
1983a.

15
Materials and Methods

2.2. Preparation of Microsomes

Rough microsomes (RM) from canine pancreas were prepared as described in Walter and
Blobel, 1983b. For preparation of puromycin-high salt treated microsomes (PKRM), a
suspension of RM containing 3.5 equivalents (eq.; for definition, see Walter, et al., 1981) per
µl was mixed with an equal volume of buffer B (100mM Hepes/KOH pH7.6, 200mM sucrose,
300mM potassium acetate, 10mM magnesium acetate, 3mM dithiothreitol (DTT), 0.2mM
GTP, protease inhibitors (10µg/ml leupeptin, 5µg/ml chymostatin, 3µg/ml elastatinal, 1µg/ml
pepstatin), 3mM puromycin). After homogenization, the mixture was incubated for 1hr at 0°C,
followed by 10 min at 37°C and 10 min at room temperature. The sample was centrifuged in a
Beckman TLA100.3 rotor for 30 min at 100,000 rpm at 2°C. The pellet was resuspended at 10
eq./µl in buffer C (50mM Hepes/KOH pH7.6, 500mM sucrose, 800mM CsCl, 15mM
magnesium acetate, 3mM DTT, protease inhibitors) and mixed with an equal volume of a
buffer identical to buffer C, except that it contained 1.95M sucrose. The total volume was
determined and additional CsCl was added to a final concentration of 700mM. The sample
was overlaid with 1ml buffer D (50mM Hepes/KOH pH7.6, 800mM sucrose, 700mM CsCl,
15mM magnesium acetate, 3mM DTT, protease inhibitors) and 0.6ml buffer B in a 13x51mm
polycarbonate tube. After centrifugation in a TLA100.3 rotor for 1hr at 100,000 rpm at 20°C,
the top 0.2ml were discarded and the following 2-2.5ml, which contained the membranes,
were collected. This fraction was diluted 1:4 in 50mM Hepes/KOH pH7.6, 1mM DTT, protease
inhibitors, and centrifuged in a TLA100.3 rotor for 30 min at 100,000 rpm at 2°C. The pellet
was resuspended in buffer A (50mM Hepes/KOH pH7.6, 250mM sucrose) containing 1mM
DTT and protease inhibitors. The membranes were washed once or twice by resuspension
and centrifugation and finally taken up at 2-3 eq./µl in buffer A containing 1mM DTT. Salt-
washed rough microsomes (KRM) were essentially prepared as PKRM, except that the
puromycin reaction was omitted and that the flotation of the membranes was carried out at
2°C.

16
Materials and Methods

2.3. Purification of Membrane Proteins and Reconstitution into


Proteoliposomes

The purification of the SRP receptor and the Sec61p complex as well as their reconstitution
into proteoliposomes were carried out as described (Görlich and Rapoport, 1993; Jungnickel
and Rapoport, 1995). The concentrations of the SRP receptor and Sec61p complex in the
suspensions of proteoliposomes were 0.8-3 eq./µl and 3-8 eq./µl, respectively.

2.4. Preparation of Ribosome-Nascent Chain Complexes

Truncated mRNAs were translated in a wheat germ or reticulocyte lysate system in the
presence of 35S-methionine (Jungnickel and Rapoport, 1995). Translation in the wheat germ
system was carried out for 13 min at 28°C, followed by addition of 2µM edeine and further
incubation for 5 min. Translation in the reticulocyte lysate was carried out for 25 min at 28°C.
Where indicated, TmD-Phe suppressor tRNA or TDBA-lysyl tRNA were present during
translation.

To isolate RNCs (see also Lauring et al., 1995a) 100µl of the translation mixture were diluted
in 900µl of 40mM Hepes/KOH pH7.6, 2mM magnesium acetate, 2mM DTT containing either
150mM potassium acetate (for low salt-washed RNCs) or 500mM potassium acetate (for high
salt-washed RNCs). The samples were layered on top of a 1ml cushion containing a low or
high salt concentration (40mM Hepes/KOH pH7.6, 0.5M sucrose, 2mM magnesium acetate,
2mM dithiothreitol and either 150mM or 500mM potassium acetate) in a 13x51mm
polycarbonate Beckman tube. After centrifugation for 1 hr at 100,000 rpm at 4°C in a
TLA100.3 rotor, the ribosome pellets were resuspended at a concentration of approximately
140nM in ribosome buffer (50mM Hepes/KOH pH7.6, 250mM sucrose, 150mM potassium
acetate, 2mM magnesium acetate).

For ribosome competition experiments, RNCs produced in a reticulocyte lysate were isolated
by centrifugation through a sucrose gradient containing 150mM potassium

17
Materials and Methods

acetate, as described for wheat germ RNCs, and incubated with 10mM N-ethylmaleimide for
40 min on ice. The reaction was quenched with 50mM DTT.

Mock translation mixtures lacking mRNA and amino acids were prepared and incubated as
described above. Salt washed, non-translating ribosomes were isolated from a mock
translation mixture as described for the isolation of salt-washed RNCs. The mock translation
was divided into a cytosolic and a ribosomal fraction by centrifugation for 1hr at 100,000 rpm
in a Beckman TLA100 rotor. The ribosome pellet was resuspended in the original volume in
ribosome buffer.

2.5. Ribosome Competition

A translation mixture containing approximately 200fmoles RNCs (determined by measuring


the absorption at 260nm and assuming that a solution with 1 A260 absorption contains 16nM
ribosomes; Hanein et al., 1996) was mixed with either a mock-translation mixture or a
translation mixture containing a different population of RNCs in which the concentration of
ribosomes was determined in the same manner. When subfractions of mock translations
were used in competition experiments, the amounts added were equivalent to the original
volume of the mock-translation mixture. Where indicated, SRP (10nM) was added after
translation and before addition of the mock-translation mix. After addition of PKRM (0.4 eq.
per 5µl final volume) or reconstituted proteoliposomes (1µl per µl of final volume) incubation
was carried out for 10 min on ice and 5 min at 28°C. Experiments with isolated RNCs were
performed in an analogous manner. Alternatively, the membranes were preincubated with
one population of ribosomes or RNCs before addition of the other population.
For competition experiments using the lepXa fragment, RNCs carrying the lepXa chain were
first targeted to the ER membrane and then subjected to treatment with factor Xa as
described in Mothes et al., 1997.

2.6. Insertion and Translocation Assays

To assay membrane insertion of ppl86, the samples were incubated with 1 volume of
1.5mg/ml proteinase K in ribosome buffer containing 1mM DTT and 8mM magnesium

18
Materials and Methods

acetate for 45 min on ice. The proteinase K reaction was stopped by precipitation of the
sample with 15% TCA.
Translocation of membrane-targeted nascent chains was induced by incubation of the
samples with 1.5mM puromycin for 10 min on ice and 30 min at 37°C.
Targeting assays using flotation of membrane-targeted RNCs in a sucrose gradient were
carried out as described (Jungnickel and Rapoport, 1993; 1995). The flotation of the
membranes was confirmed by immunoblotting with antibodies against ER membrane
proteins.
Alternatively, membrane targeting was assayed by sedimentation of the microsomes through
a sucrose cushion. After incubation of RNCs with microsomes, the sample was diluted to 30µl
and loaded on top of 200µl of a buffer containing 50mM Hepes/KOH pH7.6, 500mM sucrose,
150mM potassium acetate, 2mM magnesium acetate, 2mM DTT in 7x20mm polycarbonate
tubes (Beckman). The samples were centrifuged for 5 min at 55,000 rpm in a Beckman
TLS55 rotor at 2°C. The supernatant was precipitated with 15% TCA and both supernatant
and pellet fraction were analyzed by SDS-PAGE. Sedimentation of the membranes was
monitored by immunoblotting with antibodies against ER membrane proteins.

2.7. Sample Preparation

Most samples were precipitated with trichloroacetic acid and separated in 13.75% or 7.5-
17.5% Tris-Glycine polyacrylamide gels or in 12% or 16% Tris-Tricine gels. For experiments
using radiolabeled nascent chains, the gels were dried, exposed to Fuji PhosphoImager
screens and quantitated using the Fuji BAS1000 software. For immunoblotting, the proteins
separated by SDS-PAGE were transferred to a nitrocellulose membrane and incubated with
antibodies. Subsequently, the blots were developed using an ECL kit.

2.8. Photocrosslinking

RNCs carrying photoreactive groups were generated in the wheat germ or reticulocyte lysate
systems by translation in the presence of TmD-Phe suppressor tRNA or TDBA-lysyl tRNA.
Where indicated, the RNCs were isolated after translation.

19
Materials and Methods

After the RNCs were either targeted to ER membranes as described above or after they were
incubated with the denoted cytosolic factors for 5 min on ice and 5 min at 28°C, the samples
were irradiated for 10 min on ice. Crosslinks of ppl86 to membrane proteins were analyzed by
immunoprecipitation with Sec61α and TRAM antibodies as described (Görlich et al., 1992).

2.9. Preparation of Bovine Pancreatic Cytosol and Purification of Calmodulin

As a source of bovine cytosol, the 100,000 g supernatant of a bovine pancreatic RM


preparation was used (see Walter and Blobel, 1993). For purification of calmodulin, the
100,000 g supernatant was filtered (0.45µm pore size) and incubated for 10 min at 95°C.
Aggregates were pelleted by an initial centrifugation of the sample for 10 min at 20,800 g at
4°C and a subsequent centrifugation for 10 min at 100,000 rpm at 2°C in a Beckman
TLA100.3 rotor. The supernatant was adjusted to 50mM Hepes/KOH pH7.6, 250mM sucrose,
200mM potassium acetate, 2mM magnesium acetate, 2mM DTT and loaded onto DEAE
sepharose. After washing of the column, elution was carried out using a salt-gradient (200 to
800mM potassium acetate) in a buffer as above. All fractions and aliquots taken at various
steps of the purification procedure were tested in the crosslinking assay and analyzed by
SDS-PAGE and Coomassie staining. A protein of the expected size was present in all
fractions containing the crosslinking activity and was identified as calmodulin by sequencing.

2.10. Preparation of Ribosome-Channel Complexes for Cryo-Electron


Microscopy

mRNA coding for ppl86 was translated in rabbit reticulocyte lysate in the presence of either
PKRM or proteoliposomes containing purified SRP receptor and Sec61p complex for 20 min
at 27°C. To generate complexes lacking a nascent chain the mRNA was omitted. After
translation, 1-acyl-2-[6-{7-nitro-2,1,3,-benzoxadiazole-4-yl amino}-caproyl]-sn-glycero-3-
phosphocholine (C6-NBD-PC) in ethanol was added to a final concentration of 1mol% of total
phospholipid to follow the fractionation of

20
Materials and Methods

membranes under UV light. The translation mixture was adjusted to a final volume of 150µl
containing either 2M sucrose (PKRM) or 1.5M sucrose (proteoliposomes), both in 30mM
Hepes/KOH pH7.8 and 10mM magnesium acetate buffer. For ribosomes without or with a
nascent chain, the buffer contained 100mM or 500mM potassium acetate, respectively. The
samples were transferred to a 7x20mm polycarbonate tube (Beckman) treated with 20mg/ml
bovine serum albumin for 10 min at room temperature. Thirty µl of the same buffer without
sucrose were layered on top. For all experiments several reactions were prepared in parallel.
The sample was spun for 1h at 100,000 rpm at 2°C in a Beckman TLA100 rotor. The floated
membranes were collected using an UV trans-illuminator. They were dilute approximately 1:3
in a buffer containing 30mM Hepes/KOH pH7.8, 10mM magnesium acetate, 1.5% digitonin
(final concentration), and 100mM potassium acetate for proteoliposomes with ribosomes
lacking nascent chains or 500mM potassium acetate for proteoliposomes with ribosomes
containing nascent chains and for all PKRM samples. After incubation for 15 to 30 min at 4°C
with repeated mixing, the samples were centrifuged for 20 min at 100,000 rpm at 2°C in a
TLA100 rotor. The pellet was resuspended in 30mM Hepes/KOH pH7.8, 1.5% digitonin,
100mM potassium, and 10mM magnesium acetate. Aliquots were taken at various steps and
analyzed by SDS-PAGE and immunoblotting against the α- and β-subunits of the Sec61p
complex. Proteinase K treatment was carried out as described above and CTABr-precipitation
was done as described in Mothes et al., 1997. All samples were kept at 4°C and frozen for
electron cryo-microscopy within 4 hours of preparation.

2.11. Identification of Ribosome-Associated Membrane Proteins

Ribosome-channel complexes derived from KRM were prepared as described above, except
that after the final sedimentation the ribosome pellet was resuspended in a buffer containing
50mM HEPES/KOH pH7.8, 1200mM potassium acetate, 10mM magnesium acetate, 1.5%
digitonin, 2mM puromycin, 1mM GTP, 2mM DTT and protease inhibitors. The sample was
incubated for 30 min at 4°C and for 10 min at 37°C with repeated mixing. Then, the
ribosomes were pelleted by sedimentation for 40 min at 70,000 rpm in the TLA100.3 rotor.
The supernatant was extracted with Triton X-114 as described in Görlich et al., 1992. The
ribosome pellet was

21
Materials and Methods

resuspended in ribosome buffer. The samples were separated by SDS-PAGE and analyzed
by staining of the gel using Coomassie Brilliant Blue and by immunoblotting with antibodies
raised against Sec61α and TRAPα.

2.12. Electron Cryo-Microscopy of Ribosome-Channel Complexes

Suspensions were loaded onto air glow-discharged 300-mesh grids with thin continuous
carbon film, supported by a holey carbon mesh. The specimens were blotted and plunged
into liquid ethane (Dubochet et al., 1988) in a humid environment at 4°C (>85% relative
humidity). A Gatan cryo-transfer system and cryo-holder (model 626-DH) were used to
transfer grids into a Philips CM12 transmission electron microscope equipped with a cryo-
blade type anti-contaminator and specimen relocation system. All electron micrographs were
recorded at 100kV, under minimal dose conditions with a LaB6 filament, using a defocus
range of -1.0 to -1.5µm. Micrographs were recorded at 28,000x magnification on KODAK
SO163 film and developed for 12min in full strength D19 developer (KODAK). In some cases,
images were recorded with the specimen tilted at 30° using a dynamic defocus spot scan
package developed by Dr. I. Tews.

2.13. Three-Dimensional Image Processing and Analysis

Micrographs displaying minimal astigmatism and drift by optical diffraction were chosen for
processing. Negatives were digitized on a ZEISS SCAI scanner using a 7µm raster, binned to
14µm (corresponding to 5Å/pixel) and converted to SPIDER format. Image processing was
done using the SPIDER software package (Frank et al., 1996). In most cases, particle picking
was done by cross-correlating the image against a rotationally averaged frontal view of the
yeast ribosome. Feature with a cross-correlation peak higher than 0.5 were windowed from
the original micrographs, montaged and interactively de-selected to remove bad particles. In
difficult cases, particles were picked interactively from large sections of the original image
using WEB (Frank et al., 1996). Pre-centered 2D datasets of ribosome-channel complexes
were first aligned against the corresponding ribosome model truncated at 50Å resolution,
using Radon alignment methods (Radermacher, 1994). Three alignment

22
Materials and Methods

cycles with unshifted images were calculated by updating the 3D reference from the previous
cycle (angular refinement). Finally, the original images were shifted according to the
previously refined translations and aligned for three more cycles, allowing both angular and
translational refinement in 3D. Final 3D maps were generated using R-weighted back-
projection. The resolution in each data set was estimated as described in table 4, and is lower
than that imposed by the first node of CTF. We minimized the effects of the CTF by Fermi
low-pass filtering each final 3D map to the estimated resolution. It should be noted that a gap
between the ribosome and the channel was seen even when all frontal and similarly oriented
views, for which CTF fringes are most pronounced, were excluded from the 3D analysis.
The threshold representing 100% of the ribosomal volume was chosen on the basis of
calculated and experimentally measured partial specific volumes and the known mass of
ribosomal protein and RNA. The 100% ribosomal volume used in this work was 4x106Å3.
Statistical 3D maps were computed by randomly breaking the datasets into subsets
containing 500 particles (without image repetition), and generating the R-weighted 3D maps.
Each series of maps was used without scaling to produce an averaged 3D volume and a
second volume containing the variance. The ratio of the average to the variance was
interpreted as a measure of statistical confidence using the Student′s t-test (Tractenberg and
DeRosier, 1985).

23
Results

3. RESULTS

3.1. Calmodulin Interacts with Signal Peptides

The synthesis of proteins following the secretory pathway is initiated in the cytosol. Newly
translated signal peptides are bound by the signal recognition particle (SRP) which
subsequently assists in targeting of the ribosome-nascent chain complex to the membrane of
the endoplasmic reticulum (ER; for review, see Walter and Johnson, 1994). SRP seems to be
the most important cytosolic interaction partner of signal peptides. It has been suggested that
SRP is constantly cycling on and off ribosomes while scanning the nascent chain for a signal
sequence (Ogg and Walter, 1995). Yet, the ratio of SRP to translating ribosomes in the cell
has been estimated to be 1:10 to 1:100 (Ogg and Walter, 1995), implying that an emerging
signal sequence may not always be bound by SRP. Since the hydrophobic signal sequence
enters the hydrophilic environment of the cytosol, it seems likely that other cytosolic factors
can also interact with it. To test this hypothesis we carried out a photocrosslinking study with
translation intermediates of the secretory protein preprolactin.

Ribosome-bound translation intermediates of a defined length were generated by in vitro


translation of truncated mRNA lacking a stop codon (Perara et al., 1986; Mueckler and
Lodish, 1986). The translation products were labeled with 35S-methionine and analyzed by
SDS-PAGE.
To probe the environment of translation intermediates, modified lysines carrying a
photoreactive crosslinker were incorporated into nascent chains at positions where normally
lysines would occur. This was done by adding modified lysyl-tRNAs (TDBA-lysyl-tRNA) to the
translation reaction (Görlich et al., 1991). Upon irradiation with UV light, the crosslinker is
activated to react with molecules in the immediate vicinity (Figure 4; Kurzchalia et al., 1986;
Görlich et al., 1992a,b). Proteins in close proximity to the nascent chain were covalently
linked to the ribosome-nascent chain complex. The crosslinked product was detected by a
shift in molecular weight of the nascent chain.

24
Results

UV

AUG AUG

Figure 4: Photoreactive crosslinkers in the nascent polypeptide chain are used to probe the environment of the signal
sequence. A modified lysine carrying a photoreactive TDBA group is incorporated into the signal sequence during
translation (left). After irradiation with UV light, the nascent chain is covalently linked to proteins (shown in red) in
close proximity to the signal sequence (right). Due to an increased molecular weight, the crosslinking product can be
detected after the sample has been separated by SDS-PAGE.

When microsomes were added to translation intermediates, the ribosomes were bound to the
membrane and the nascent chain was able to interact with the translocation channel (Perara
et al., 1986; Connolly et al., 1989). As a result, stable translocation intermediates were
created representing distinct stages of protein translocation across the ER membrane
depending on the length of the nascent chain.

In order to probe the cytosolic environment of signal peptides, translation intermediates of


preprolactin comprising the N-terminal 86 amino acids (ppl86) were generated in a wheat
germ system containing very little endogenous SRP. Ribosome-bound translation products
were separated from the translation reaction by sedimentation through a sucrose cushion
(Jungnickel and Rapoport, 1995). The ppl86 chain has lysines with photoreactive crosslinkers
in positions 4, 9, 72 and 78. Since amino acids 72 and 78 are buried within the ribosome, only
the two lysine residues in the signal sequence, K4 and K9, could give rise to crosslinks to
potential cytosolic interaction partners (Kurzchalia et al., 1986).
When wheat germ cytosol was readded to the translation intermediates, a major crosslink
appeared after UV irradiation (Figure 5, lane 3, marked by a star). The interacting partner had
an approximate molecular weight of 17kDa. In the absence of cytosol or without irradiation no
crosslink was seen (Figure 5, lanes 1 and 2).
A mutant ppl86 chain that has three hydrophobic amino acids deleted from its signal
sequence (ppl86∆13-15; Jungnickel and Rapoport, 1995) did not give rise to a

25
Results

crosslink to the 17kDa protein (data not shown). This suggests that the interaction with the
17kDa protein depends on a functional signal sequence.

Figure 5: Crosslinking of the ppl86 chain to a cytosolic protein of about 17kDa. Ribosomes carrying the first 86 amino
acids of the secretory protein preprolactin were generated in a wheat germ translation system. After translation, the
RNCs were isolated by centrifugation through a sucrose cushion. The isolated RNCs were incubated with the
following components: wheat germ extract (WG, lanes 3, 5, 7, 9, 11, 13, 15 and 17); purified nascent polypeptide-
associated complex (NAC, lanes 4, 5, 8, 9, 12, 13, 16 and 17); microsomes (PKRM, lanes 6 to 9 and lanes 14 to 17);
purified SRP (SRP, lanes 10 to 17). After incubation, the samples were subjected to UV irradiation and analyzed by
SDS-PAGE. ppl86 x SRP54 indicates crosslinks of the nascent chain to the 54kDa subunit of SRP; ppl86 x
Sec61p/TRAM points to crosslinks of ppl86 to the Sec61p complex and the TRAM protein. Lane 1 shows a control
sample without irradiation, lane 2 shows a sample that was incubated in the absence of additional factors.

Next, we tested whether signal sequence binding by the 17kDa protein can be competed by
other factors. The nascent polypeptide-associated complex (NAC) has been reported to
interact with both ribosomes (Möller et al., 1998) and with ribosome-nascent chain complexes
(RNC) with or without a signal sequence (Wiedmann et al., 1994). When purified NAC was
added to isolated ppl86 translation intermediates, no crosslinks were seen (Figure 5, lane 4).
In the presence of both purified NAC and wheat germ cytosol, the 17kDa protein remained
bound to the nascent chain (lane 5). In contrast, the intensity of the crosslink of ppl86 to the
17kDa protein was reduced dramatically when purified SRP was added (lane 11 vs. lane 3).
Instead, a crosslink to the 54kDa subunit of SRP appeared (lane 11, marked ppl86 x SRP54).
When microsomes were present (Figure 5, lanes 6 to 9 and 14 to

26
Results

17), both the crosslinks to the unknown 17kDa protein (lane 7 vs. lane 3) and to SRP54
(lanes 14 to 17 vs. lanes 10 to 13) disappeared. Instead, crosslinking products to components
of the translocation machinery were seen (marked ppl86 x Sec61/TRAM; see also Jungnickel
and Rapoport, 1995), suggesting that the nascent chain inserted properly into the
translocation channel.
These data indicate that the interaction of the 17kDa protein with the signal peptide can be
competed by SRP and ER membranes.

We then used ion exchange chromatography to identify the binding partner of the preprolactin
signal sequence. Since a crosslink to a protein of similar size was also seen with other
cytosolic extracts (see below) we chose bovine pancreas as a source of cytosol for
purification of the crosslinking activity. Using the photocrosslinking assay to monitor the
purification process, we isolated the 17kDa protein in a simple two step procedure:
1. Incubation of the cytosol at 95°C and sedimentation of aggregated material
The 17kDa protein proved to be very thermostable and remained soluble (data not shown).
2. Ion-exchange chromatography using DEAE sepharose
The crosslinking activity was bound to the column in the presence of 200mM potassium
acetate (data not shown). Elution was carried out with a potassium acetate gradient ranging
from 200 to 800mM and fractions were collected. Starting at a concentration of about 500mM
potassium acetate, a protein of the expected size was eluted from the DEAE sepharose
(Figure 6A, fraction 9 to 12; marked by a star). All fractions containing this protein showed a
high crosslinking activity, indicating that it was indeed the interaction partner of the signal
sequence (Figure 6B, lanes 8 to 11; the crosslinked product is indicated by a star).

The protein was sequenced and identified as calmodulin. We confirmed that calmodulin can
bind signal sequences by testing commercially available calmodulin (purified from bovine
brain) in the photocrosslinking assay (Figure 7). A crosslink of the expected size was visible
(marked ppl86 x CaM) in the presence of either bovine brain calmodulin (lane 3), wheat germ
extract (lane 4), bovine pancreatic cytosol (lane 5) or calmodulin purified from this source
(lane 6).

27
Results

Figure 6: Purification of calmodulin. A: Cytosolic extract from bovine pancreas was boiled for 10 min at 95°C and
aggregates were removed by centrifugation. Proteins in the supernatant were bound to DEAE-sepharose and, after
washing of the column, were eluted with a potassium acetate gradient ranging from 200mM to 800mM. Aliquots of the
collected fractions (1 to 18), of load, flow through (FT) and wash were precipitated with 15% TCA, separated by SDS-
PAGE and analyzed by staining with Coomassie Brilliant Blue. The star indicates the protein that was identified as
calmodulin. B: Aliquots of DEAE-elution fractions, of load, flow through (FT) and wash were tested for crosslinking
activity using the 86mer of ppl. The star denotes the position of the crosslink to calmodulin; ppl86 marks the position
of the nascent chain.

28
Results

Figure 7: Calmodulin from different sources binds to the signal sequence of ppl86. RNCs of ppl86 were produced in a
wheat germ system and isolated by sedimentation through a sucrose cushion. After isolation, the sample was
supplemented with either commercially available calmodulin purified from bovine brain (CaM-BB, lane 3), wheat germ
extract (WG, lane 4), bovine pancreatic extract (BPE, lane 5) or calmodulin purified from a bovine pancreatic extract
(CaM-BPE, lane 6). Subsequently, the samples were irradiated with UV light and analyzed by SDS-PAGE. ppl86 x
CaM indicates crosslinks of the ppl86 chain to calmodulin. Samples not subjected to irradiation or incubated in the
absence of additional factors are shown in lanes 1 and 2, respectively.

2+
Figure 8: Calmodulin binding to the signal sequence of preprolactin is dependent on Ca and sensitive to calmodulin
inhibitors. In a wheat germ translation system, RNCs of ppl86 were assembled and subjected to UV irradiation after
incubation with a variety of factors. A non-irradiated control sample is shown in lane 1. 1mM CaCl2 was added to all
samples except for samples 1 and 2 (lanes 1 and 2). Samples depicted in lanes 3 to 5 were supplemented with
increasing concentrations of calmidazolium chloride (CAM), whereas increasing amounts of trifluoperazine (TFP)
2+
were added to the samples shown in lanes 8 to 10. In addition, 1mM or 5mM EGTA were used to deplete Ca (lanes
6 and 7, respectively). SDS-PAGE was used to separate crosslinked products and nascent chains that had not been
linked to other proteins. Crosslinks of ppl86 to calmodulin are indicated by ppl86 x CaM, crosslinks to other cytosolic
factors by ppl86 x NAC and ppl86 x SRP.

29
Results

It has been shown that substrate binding by calmodulin is dependent on Ca2+ (James et al.,
1995) and can be repressed by specific inhibitors. Therefore, we studied the effect of two
frequently used calmodulin inhibitors (calmidazolium chloride and trifluoperazine; Cook et al.,
1994; Uemura and Taketomi, 1995) and of the Ca2+ chelator EGTA on signal sequence
binding by calmodulin. For this and the following experiments, crosslinking was carried out
with RNCs that had not been isolated by sedimentation through a sucrose cushion. Instead,
the wheat germ translation system, which had been used to generate the RNCs, was
supplemented with additional factors after translation.
Upon UV irradiation in the full translation system (Figure 8, lane 2 vs. lane 1), a weaker
crosslink to calmodulin was seen (marked ppl86 x CaM). Furthermore, crosslinks to NAC
(marked ppl86 x NAC) and residual wheat germ SRP (marked ppl86 x SRP54) were
detected. Addition of 1mM Ca2+ dramatically increased the amount of nascent chain
interacting with calmodulin (lane 11 vs. lane 2). The presence of 1mM EGTA did not have a
pronounced effect (lane 6). However, when 5mM EGTA were added, the crosslinks to
calmodulin disappeared completely (lane 7). This suggests that calmodulin binding to the
signal sequence depends on Ca2+. Both calmodulin inhibitors tested, calmidazolium chloride
(Figure 8, CAM) and trifluoperazine (Figure 8, TFP), also prevented the interaction of
calmodulin with the signal sequence of preprolactin (CAM, lanes 3 to 5 and TFP, lanes 8 to
10). The addition of DMSO, the solvent of the inhibitors, did not have an effect (Figure 8, lane
12).

For SRP it has been shown that chain elongation renders the signal sequence incompetent
for binding to SRP54 (Siegel and Walter, 1988). This is probably due to increased folding of
the nascent chain and reflects a translocation-incompetent stage of ribosome-nascent chain
complexes. Using longer translation intermediates of preprolactin we tested whether the
calmodulin/signal sequence interaction was also dependent on the length of the nascent
chain. Indeed, fragments of preprolactin with the N-terminal 132 amino acids showed a
reduced crosslinking efficiency to calmodulin when compared to ppl86 (data not shown) and
calmodulin binding to a preprolactin chain with 169 amino acids was almost not detectable
(data not shown). Thus, binding of calmodulin is decreased upon chain elongation.

30
Results

To verify that the interaction of signal peptides with calmodulin is not limited to preprolactin
we tested other substrates. Using the photocrosslinking approach, calmodulin binding was
demonstrated for both the signal sequence of the secretory protein preinvertase and the
signal anchor of the integral membrane protein leader peptidase (data not shown).
On the other hand, no interaction was seen using an 86 amino acid fragment of the secretory
protein preproαfactor (ppαF86) with a single modified lysine in position 5. However,
calmodulin binding to ppαF86 was readily detectable when the environment of the
hydrophobic core of the ppαF signal peptide was tested directly using a different site specific
photocrosslinking technique (High et al., 1993; Martoglio et al., 1995). In this approach we
employed truncated mRNA with an amber stop codon at the position where a crosslinker was
desired. During in vitro translation amber tRNA carrying a modified phenylalanine with a
photoreactive crosslinker was present. Addition of the modified amber tRNA simultaneously
suppressed the termination signal and incorporated a photoreactive group into the nascent
chain.

Figure 9: Crosslinking of ppαF86 to calmodulin. Polypeptides corresponding to the 86 amino terminal residues of
preproαfactor (ppαF86) were synthesized in wheat germ extract in the presence of amber tRNA carrying a modified
phenylalanine with a photoreactive group. The photocrosslinker was placed either in position 15 (amb15, lanes 9 to
16) or in position 16 (amb16, lanes 1 to 8) of the ppαF signal sequence. After translation, samples were
supplemented with either 1mM CaCl2 (lanes 4 to 8 and 12 to 16), 5mM EGTA (lanes 5, 7, 13 and 15), purified
calmodulin (CaM, lanes 6, 7, 14 and 15) or purified SRP (lanes 8 and 16). The crosslink of the nascent chain to
calmodulin is marked as ppαF86 x CaM. ppαF86 x SRP54 indicates the crosslink to the 54kDa subunit of SRP.
Lanes 1 and 9 are controls without suppressor-tRNA, lanes 2 and 10 show samples that were not subjected to UV
irradiation.

31
Results

Using the suppressor tRNA technique, it was shown that both the crosslinking efficiency and
the electrophoretic mobility of the crosslink to calmodulin were clearly dependent on the
position of the photoreactive group. Representative results for ppαF86 with a
photocrosslinker in position 15 or 16 are presented in Figure 9 (amb15 and amb16,
respectively). In addition, translation intermediates of ppαF86 with crosslinkers either in
position 8, 9, 12, 13 or 14 were tested (data not shown).
For both ppαF86amb15 and ppαF86amb16, a fragment corresponding to the first 86 amino
acids of preproαfactor appeared upon addition of the amber tRNA to the translation reaction
(Figure 9, lanes 2 and 10 vs. lanes 1 and 9, marked ppαF86) indicating that the stop codon
was suppressed. When the sample was exposed to UV light and Ca2+ had been added, a
crosslink to calmodulin was apparent for ppαF86amb16 (Figure 9, lane 4, indicated by
ppαF86 x CaM) but only a very weak interaction was detected in the case of ppαF86 amb15
(Figure 9, lane 12). When the reaction was supplemented with additional pure calmodulin, the
crosslinking efficiency was increased for ppαF86amb15 (lane 14 vs. lane 12) but remained
about the same for ppαF86amb16 (lane 6 vs. lane 4). The crosslink disappeared upon
addition of EGTA (lanes 5, 7, 13 and 15), confirming that the binding partner was indeed
calmodulin. As expected, in the presence of purified SRP, crosslinks to the 54kDa subunit
were visible (lanes 8 and 16, marked by ppαF x SRP54).
Based on the results obtained with preproαfactor, preinvertase and leader peptidase, we
conclude that the ability to bind to calmodulin is a general feature of signal sequences. These
results suggest that in addition to SRP, calmodulin may be involved in the recognition of
signal sequences.

32
Results

3.2. Regulation of Ribosome Binding to the ER Membrane

It has been shown that all ribosomes, regardless of the presence and nature of a nascent
chain can bind to the Sec61p complex (Borgese et al., 1974; Kalies et al., 1994; Lauring et
al., 1995b; Jungnickel and Rapoport, 1995). This raises the question of how efficient targeting
of RNCs carrying signal sequences can occur.
It has been suggested that the cytosolic factor NAC (nascent polypeptide-associated
complex) is an inhibitor of unspecific interactions of ribosomes and RNCs with SRP and the
ER membrane (Wiedmann et al., 1994; Lauring et al., 1995a,b; Möller et al., 1998). Binding of
SRP to ribosomes synthesizing nascent chains containing signal sequences would compete
off NAC, thereby allowing RNCs with a signal sequence to bind to the ER membrane and
engage in protein translocation.

However, the role of NAC in the regulation of ribosome targeting to the ER membrane is
controversial (see Introduction and Powers and Walter, 1996). Therefore, we decided to
reevaluate the function of NAC and the role of SRP in achieving specific targeting of
ribosome-nascent chain complexes carrying a signal sequence.

3.2.1. NAC Does Not Prevent Ribosome Binding to the ER Membrane

In previous experiments it has been demonstrated that high salt washed RNCs can be
targeted to the ER membrane in an SRP-independent manner (Jungnickel and Rapoport,
1995; Lauring et al., 1995a,b). This was assumed to be due to the removal of NAC by the
high salt treatment. We now performed targeting assays in a complete translation system to
test whether SRP-independent targeting can occur in the presence of NAC and other
cytosolic factors.

To assay SRP-independent targeting of RNCs, a wheat germ system containing low levels of
endogenous SRP was used for generation of translation intermediates. Furthermore, wheat
germ SRP is known to interact poorly with canine SRP receptor (Prehn et al., 1987). SRP
was also absent from the microsomes used in targeting assays because the membranes had
been treated with puromycin and high salt

33
Results

concentrations (PKRM=puromycin/high salt treated microsomes). This procedure not only


removes endogenous ribosomes and RNCs but also endogenous SRP.
In addition, we wished to confirm that the wheat germ extract employed here contained
physiological concentrations of NAC. Therefore, we compared the amount of NAC in 5
different wheat germ extracts by immunoblotting with antibodies against mammalian NACα
(Wiedmann et al., 1994; data not shown). The concentration of NAC in the extracts differed
by no more than a factor of three, with the wheat germ extract used for targeting assays
containing an intermediate concentration. The absolute concentration of NAC was calculated
to be about 0.8µM by comparing it with recombinant mammalian NAC, approximately the
same as in reticulocyte lysate. Thus, we conclude that our system is comparable to the
system used for establishing the NAC model (Wiedmann et al., 1994; Lauring et al., 1995a,b).

For targeting assays, RNCs carrying the ppl86 fragment were first generated in the wheat
germ system, then canine pancreatic microsomes were added. Targeting that has been
uncoupled from translation is similar to targeting in a truly cotranslational system (Perara et
al., 1986), yet ribosome binding is more efficient since conditions for targeting can be
optimized without affecting translation. By using PKRM in our experiments, we also increased
the number of available ribosome binding sites.
Targeting of RNCs carrying the ppl86 chain in the absence of SRP, but in the presence of
NAC and other cytosolic factors was assessed using three independent assays. To evaluate
the efficiency of SRP-independent targeting we carried out parallel experiments using RNCs
that had been incubated with purified SRP after translation but before the addition of
microsomes.

First, a photocrosslinking experiment using TDBA-lysine was performed to probe the


environment of the ppl86 chain after addition of microsomes. RNCs carrying ppl86 have been
previously shown to represent a stage of translocation at which the nascent chain is inserted
into the translocation channel and the signal sequence is bound by the Sec61p complex
(Görlich et al., 1992a,b; Jungnickel and Rapoport, 1995).
In the absence of added SRP, crosslinks of ppl86 to Sec61α and TRAM were observed when
PKRM were added (Figure 10, lane 3 vs. lane 2, indicated by ppl86 x

34
Results

Sec61α/TRAM). The identity of the crosslinked products was confirmed by


immunoprecipitations (Figure 10, lane 6 and 7). This shows that even in the presence of NAC
and other cytosolic factors, the signal sequence of the nascent chain can contact the
translocation channel, indicating that the RNCs are efficiently targeted to the ER.
Crosslinks of the nascent chain to the 54 kDa subunit of SRP were visible when SRP was
added (Figure 10, lane 4 and with reduced intensity in lane 5, marked by ppl86 x SRP54)
confirming that indeed the signal sequence had been bound by SRP.
In the presence of SRP and microsomes membrane crosslinks appeared, which were similar
to those observed in the absence of SRP (Figure 10, lanes 5, 9 and 10). These data suggest
that SRP is not essential for efficient targeting of RNCs carrying ppl86 in the presence of NAC
and other cytosolic factors

A second approach confirmed these results. We directly tested binding of RNCs to


microsomes by employing a flotation assay. RNCs carrying ppl86 were synthesized in wheat
germ extract and incubated with PKRM. Next, the membranes were subjected to flotation in a
sucrose gradient at physiological salt concentrations. More than 80% of the nascent chain
was detected in the membrane fraction both in the absence and presence of SRP (data not
shown). Again, these data show that even in the presence of NAC and other cytosolic factors,
RNCs can bind to ER membranes in an SRP-independent manner.
To test whether the nascent chain had been inserted into the translocation channel and
whether the signal sequence had been bound by the Sec61p complex, the flotation was
repeated under high salt conditions (Jungnickel and Rapoport, 1995). Again, more than 80%
of the ppl86 chain was floated with the membranes both in the presence and absence of SRP
(data not shown). This confirms that for RNCs carrying ppl86, the transition from loose to tight
membrane binding can occur independently of SRP even in the presence of NAC and other
cytosolic factors.

35
Results

36
Results

We verified our observations using another targeting assay in which the accessibility of
nascent chains to protease is determined. Nascent chains whose signal sequence has been
bound by the Sec61p complex become protected against proteinase K added from the
cytosolic side.
When ppl86 chains synthesized in wheat germ extract were treated with proteinase K in the
absence of microsomes, a small fragment of about 30 amino acids was produced (Figure 11,
lane 2, marked by a star), representing the C-terminal portion of the nascent chain that is
protected by the ribosome. In the presence of membranes, the small fragment largely
disappeared and the protected ppl86 chain appeared (Figure 11, indicated by ppl86, compare
lanes 3 and 2; for quantitation of targeting efficiency we determined the amount of
radioactivity in the ppl86 fragment compared to total protease-protected radioactivity in the
sample).
To prove that the protease-protected nascent chains represent translocation intermediates,
the polypeptides were released from the ribosome by treatment with puromycin. Properly
inserted nascent chains resume translocation after puromycin release; they move into the ER
lumen and undergo signal sequence cleavage. Indeed, puromycin-induced signal sequence
cleavage was observed (Figure 11, lane 6; ppl86-sp indicates the signal sequence cleaved
fragment). The processed ppl86 chains were protected against digestion by proteinase K in
the absence of detergent (Figure 11, lane 9) but not in the presence of detergent (Figure 11,
lane 12) indicating that the polypeptide had indeed been translocated into the lumen of the
vesicles.
In parallel, samples were analyzed that had been supplemented with purified SRP before
addition of microsomes (Figure 11, lanes 4, 7, 10 and 13). Quantitation of the results showed
that the efficiency of targeting and translocation were identical in the absence and presence
of SRP (see Figure 11).

Taken together, these results show that efficient SRP-independent targeting of RNCs and
translocation of the nascent chain can occur even in the presence of NAC and other cytosolic
factors. Thus, we conclude that NAC does not inhibit ribosome and RNC binding to the ER
membrane and therefore, that NAC does not have a function in achieving specificity of
targeting.

37
Results

3.2.2. RNCs Carrying a Signal Sequence Compete with Nontranslating


Ribosomes for Membrane-Binding Sites

In addition to challenging the role of NAC in regulation of ribosome binding to the ER


membrane, our data also contradict the general view that SRP is essential for membrane
targeting of RNCs carrying signal sequences (Walter and Blobel, 1980). This discrepancy
might be explained by two major differences between our system and the one used in earlier
studies, which have shown that RNCs are targeted to the ER in an SRP-dependent manner.
First, earlier data were derived from experiments employing rough microsomes (RM) or salt-
washed microsomes (KRM) which contained endogenous ribosomes. Instead, we used
microsomes that had been treated with puromycin under high salt conditions to remove
endogenous ribosomes and RNCs (PKRM). Thus, in PKRM there are more unoccupied
ribosome binding sites available than in the previously used microsomes.
Second, the wheat germ extract used for the targeting assays might show an increased
translation efficiency compared to wheat germ extracts used in earlier studies, resulting in a
higher fraction of RNCs in the total number of ribosomes. If one assumes that in the absence
of SRP all ribosomes, including RNCs carrying a signal sequence, compete for common
membrane binding sites, an increased translation efficiency would result in increased
targeting efficiency of RNCs. Likewise, a higher number of available ribosome binding sites,
such as the ones found in PKRM, would allow more efficient targeting of RNCs in the
absence of SRP.
The use of truncated nascent chains in our targeting reaction is not likely to account for the
discrepancy in SRP dependence because SRP has previously been found to be required for
targeting of these chains (Connolly and Gilmore, 1986).

We wanted to test the assumption that in the absence of SRP, nontranslating ribosomes and
RNCs carrying a signal sequence compete for binding to the ER membrane. In addition, we
wished to evaluate the role of SRP in the competition. To this end, we simulated a system
with low translation efficiency.
As before, RNCs carrying the ppl86 fragment were generated in the wheat germ extract
containing low levels of SRP. Next, the RNCs were incubated with PKRM in the presence of
increasing amounts of a non-programmed translation mixture (lacking

38
Results

mRNA and amino acids; mock translation) that was prepared using the same wheat germ
extract. Binding of RNCs and insertion of the ppl86 chain was assessed in a protease
protection assay (Figure 12). In the presence of mock translation mixture, SRP-independent
targeting of ppl86 was indeed greatly reduced. This was the case regardless of whether the
membranes had been preincubated with the mock translation (Figure 12, compare lanes 4 to
7 to lane 3; ppl86 marks the position of the ppl86 nascent chain, the star denotes the 30
amino acid fragment) or whether the mock and ppl86 translation reactions were added to the
membranes at the same time (data not shown). Thus, low translation efficiency indeed seems
to reduce SRP-independent membrane targeting of RNC. This is consistent with the idea that
in the absence of SRP, non-translating ribosomes can compete with RNCs for membrane
binding sites. Interestingly, the addition of SRP restored efficient targeting of RNCs carrying a
signal sequence even when a large excess of non-programmed translation mixture was
present (Figure 12, lanes 8 to 12) suggesting that binding of SRP gives RNCs with a signal
sequence a competitive advantage over nontranslating ribosomes.

39
Results

Next, we tested whether providing additional binding sites can rescue SRP-independent
targeting in a system with low translation efficiency. We performed a protease-protection
experiment with the 86mer of preprolactin and increasing amounts of PKRM. Targeting of
RNCs carrying ppl86 was examined in the absence and presence of a sixfold excess of mock
translation mixture. In the absence of mock translation at all membrane concentrations, a
substantial part of the nascent chains was resistant to protease treatment, even without
added SRP (Figure 13, lane 3 to 5). When mock translation mixture was added, barely any
targeting of RNCs was observed in the sample with the lowest membrane concentration
(Figure 13, lane 6) whereas almost complete targeting occurred at the highest concentration
(Figure 13, lane 8). This indicates that the number of available binding sites indeed
determines the extent of SRP-independent targeting in a system with low translation
efficiency. Again, addition of SRP restored efficient targeting of RNCs carrying a signal
sequence (Figure 13, lanes 12 to 14). As before, these data support the idea that in the
absence of SRP, nontranslating ribosomes compete with RNCs for common membrane
binding sites, but that RNCs carrying a signal sequence with bound SRP are given an
advantage over other ribosomes.

40
Results

Next, we wished to confirm that nontranslating ribosomes are the cytosolic factors that inhibit
SRP-independent targeting of RNCs. Thus, we separated the mock translation mixture into a
ribosome pellet and a cytosolic supernatant. As expected, SRP-independent targeting of
ppl86 synthesized in wheat germ extract was much reduced when either a sixfold excess of
mock translation mixture (Figure 14, lane 4 vs. lane 3) or an equivalent amount of isolated
ribosomes were added (Figure 14, lane 6). However, targeting efficiency was not affected by
the addition of the cytosolic supernatant (Figure 14, lane 5). In the presence of both the
supernatant and pellet fractions, inhibition of SRP-independent targeting was as pronounced
as with the complete mock translation mixture (Figure 14, lane 7 vs. lane 4). These data
demonstrate that indeed nontranslating ribosomes, but not other cytosolic factors, inhibit
SRP-independent targeting of RNCs carrying a signal sequence. Again, the addition of SRP
(Figure 14, lanes 9 to 12) allowed efficient targeting of ppl86 even when competing
nontranslating ribosomes were present (Figure 14, lanes 9, 11 and 12).

Figure 14: Inhibition of ppl86 targeting by ribosomes. RNCs of ppl86 were assembled in wheat germ extract. Where
indicated, a sixfold excess of mock translation mixture was added, or the mock translation was separated into a
ribosome pellet and a cytosolic supernatant and equivalent amounts of these fractions were added. Then, the RNCs
were bound to PKRM in the absence (lanes 3 to 7) or presence (lanes 8 to 12) of SRP. After targeting, the samples
were digested with proteinase K (the amount of protease-protected material is given as % protection). Lane 1 shows
undigested ppl86 (total), in lane 2 a sample of protease-treated ppl86 in the absence of microsomes is presented.
The star indicates the position of the ribosome-protected fragment of about 30 residues; ppl86 marks the ppl86 chain.

We also performed competition experiments using RNCs carrying ppl86 that had been
generated in wheat germ extract and then had been isolated through a sucrose cushion at
either physiological salt concentration (150mM, low salt RNCs) or high salt concentration
(500mM, high salt RNCs). By doing these experiments, we again

41
Results

examined the role of NAC in regulation of ribosome targeting. Low and high salt washed
RNCs had previously been used to establish the NAC model. Under high salt conditions,
ribosome-associated proteins such as NAC, are depleted from the RNCs (Wiedmann et al.,
1994), whereas NAC remains bound to RNCs washed at low salt. The presence of NAC in
the low salt washed RNCs and its absence from high salt washed RNCs was confirmed by
photocrosslinking experiments using RNCs carrying a fragment of 77 amino acids of firefly
luciferase (data not shown; see also Wiedmann et al., 1994). Despite the fact that low and
high salt RNCs differed in the amount of associated NAC they behaved identically in the
targeting assay. A mock translation mixture as well as the ribosome portion of a fractionated
mock translation inhibited binding of RNCs to the membrane (data not shown). Addition of the
cytosolic supernatant, even though it contained a large amount of NAC, did not affect
targeting (data not shown). High salt washed isolated ribosomes also had a strong inhibitory
effect (data not shown).

Taken together, these results show that in the absence of SRP, RNCs carrying a signal
sequence compete with nontranslating ribosomes for binding to the ER membrane. However,
the interaction with SRP gives RNCs with a signal sequence a competitive advantage over
other ribosomes. As shown before, NAC, or other cytosolic factors, does not seem to inhibit
ribosome binding to the membrane. Based on these conclusions we now understand why
efficient targeting in the absence of SRP was observed in the experiments presented in
Figure 10 and 11; we had supplied sufficient membrane binding sites for both nontranslating
ribosomes and RNCs.

3.2.3. SRP-Independent Targeting in the Reticulocyte Lysate System

We wished to exclude the possibility that our observations on SRP-independent targeting and
the lack of inhibition by NAC were restricted to the heterologous system used so far
consisting of RNCs produced in wheat germ extract and of canine microsomes. Therefore, we
performed targeting reactions with RNCs carrying ppl86 generated in the reticulocyte lysate
translation system. Rabbit reticulocyte lysate contains endogenous SRP that interacts well
with canine microsomes (Meyer et al., 1982).

42
Results

After translation in the reticulocyte lysate system, RNCs carrying ppl86 were isolated by
sedimentation through a sucrose cushion under physiological salt conditions.
Photocrosslinking experiments have shown that under these conditions SRP remains bound
to RNCs carrying ppl86 (data not shown). The presence of NAC in RNCs isolated under
these conditions was confirmed by photocrosslinking with a 77 amino acid fragment of firefly
luciferase (data not shown). The isolated ppl86 RNCs were then targeted to microsomes and
subjected to treatment with proteinase K. In the absence of microsomes, most of the nascent
chains were degraded to the 30 amino acid fragment that corresponds to the C-terminal
portion of the nascent chain buried within the ribosome (Figure 15, lane 2). A small portion of
the nascent chain, however, gave a fragment only slightly smaller than ppl86 (lane 2,
indicated by an arrow). This may be due to partial protection of the nascent chain by a
cytosolic protein. In the presence of microsomes most of the ppl86 was fully protected against
proteinase K (Figure 15, lane 3). The addition of competing nontranslating ribosomes did not
prevent targeting of RNCs (Figure 15, lanes 4 to 6) as observed before in the wheat germ
system in the presence of SRP (see Figure 12).

Figure 15: SRP-independent targeting in the reticulocyte lysate system. ppl86 was synthesized in a rabbit reticulocyte
lysate translation system and the assembled RNCs were isolated by sedimentation through a sucrose cushion under
low salt conditions. One half of the sample was treated with NEM to inactivate SRP, the other half remained
untreated. Before incubation of the RNC with PKRM, low salt washed reticulocyte ribosomes, which had also been
treated with NEM, were added in increasing amounts as indicated (given as fold excess over RNCs). Efficiency of
membrane targeting was tested by treatment with proteinase K and is given as % protection. In lanes 1 and 7
undigested samples are shown (total). Lanes 2 and 8 present samples that were treated with proteinase K in the
absence of microsomes (arrows indicate a fragment slightly smaller than ppl86 that is presumably protected from
proteolysis by a cytosolic protein). The star marks the position of the 30 amino acid fragment protected by the
ribosome; ppl86 indicates the position of the ppl86 chain.

43
Results

To test for SRP-independent targeting we treated the isolated RNCs with N-ethylmaleimide
(NEM) to inactivate SRP. It is known that membrane binding of ribosomes (Bacher et al.,
1996) and the release of the nascent chain by puromycin (data not shown) are not affected by
treatment with NEM. The function of NAC is also predicted to be insensitive to NEM-treatment
because both subunits of NAC lack cysteines (Kanno et al., 1992; Yotov and St-Arnaud,
1996). As expected, the NEM-treated RNCs were still targeted to the membrane (Figure 15,
lane 9). However, the efficiency of their membrane binding was much reduced in the
presence of competing nontranslating ribosomes (Figure 15, lanes 10 to 12).

These results show that SRP-independent targeting in the presence of NAC and other
cytosolic factors is not restricted to the wheat germ translation system. They provide further
evidence for a model where in the absence of SRP, RNCs compete with nontranslating
ribosomes for common binding sites but are given a competitive advantage over these
ribosomes in the presence of SRP.

3.2.4. SRP-Independent Targeting to Reconstituted Proteoliposomes

To approach the mechanism by which SRP confers an advantage to RNCs in their


competition with nontranslating ribosomes, we performed targeting assays with reconstituted
proteoliposomes containing only the SRP receptor and the Sec61p complex purified from
canine rough microsomes. RNCs carrying ppl86 were generated in a wheat germ extract and
bound to Sec61p complex in proteoliposomes in the presence or absence of competing
ribosomes. The targeting efficiency was assayed by proteinase K-treatment as discussed
before.
As with native microsomes, in the absence of SRP, targeting was greatly reduced when
competing nontranslating ribosomes were added (Figure 16, compare lanes 4 to 6 to lane 3).
Binding of SRP allowed efficient targeting of RNCs even in the presence of competing
ribosomes (Figure 16, lanes 7 to 10).

44
Results

Figure 16: Targeting of ppl86 to reconstituted proteoliposomes. RNCs of ppl86 generated in a wheat germ system
were incubated with reconstituted proteoliposomes containing purified SRP receptor (SR) and Sec61p complex
(Sec61p) in the absence (lanes 3 to 6) or presence (lanes7 to 10) of SRP. Where indicated, mock translation mixture
was added (given as fold excess over RNCs). Membrane targeting was tested using the protease protection assay.
Lane 1 shows an undigested sample (total), all other samples were treated with proteinase K. ppl86 denotes the
ppl86 chain. The star marks the position of the ribosome protected fragment of about 30 residues.

When proteoliposomes containing only the Sec61p complex were used in the targeting assay,
the RNCs carrying ppl86 were targeted to the membrane independently of SRP. As before,
upon addition of competing ribosomes SRP-independent targeting was inhibited (data not
shown). In the presence of SRP, very little membrane binding of RNCs occurred (<5%, data
not shown) and was completely abolished when competing ribosomes were added (data not
shown). Presumably SRP remains bound to the signal sequence in the absence of SRP
receptor and prevents insertion of the nascent chain into the translocation channel.
These results show that RNCs and nontranslating ribosomes compete for binding to the
Sec61p complex. Furthermore, the binding of SRP to the signal sequence and to SRP
receptor in conjunction with the interaction of RNC and Sec61p complex are necessary and
sufficient to overcome the competition by nontranslating ribosomes.

45
Results

3.2.5. Binding of the Signal Sequence to the Sec61p Complex Is Necessary


for Gaining a Competitive Advantage

We then wanted to clarify the significance of the interaction between RNCs carrying a signal
sequence and the Sec61p complex for the SRP-mediated competitive advantage. Thus, we
asked whether binding of the signal sequence-bearing ribosome to the Sec61p complex is
sufficient for gaining an SRP advantage or whether the signal sequence needs to be
recognized by the Sec61p complex. To this end, we carried out competition experiments with
a mutant ppl chain lacking 3 leucines within the hydrophobic core of the signal sequence
(ppl∆13-15; Jungnickel and Rapoport, 1995). The mutant signal sequence binds to SRP,
albeit with a 30% reduced efficiency compared to wild type preprolactin (judged by
photocrosslinking; Jungnickel and Rapoport, 1995). However, the signal sequence is not
bound by the Sec61p complex and therefore is non-functional for translocation. RNCs
carrying the ppl86 fragment are targeted to the membrane but the transition from loose to
tight ribosome binding to the Sec61p complex does not take place and the full length protein
bearing the deletion is translocated across the ER membrane only with a low efficiency (0.5-
2.5%; Jungnickel and Rapoport, 1995). When membrane-bound RNCs carrying ppl86∆13-15
were subjected to treatment with proteinase K, both in the absence and presence of SRP, the
majority of the nascent chains was protected (data not shown). However, a fraction of
ppl86∆13-15 was also degraded to a fragment of about 50 amino acids (data not shown). In
the presence of competing nontranslating ribosomes, the 86 amino acid and the 50 amino
acid fragments disappeared and the ribosome-protected 30 amino acid fragment became
more prominent (data not shown). Ribosome competition was equally effective in samples
with and without SRP (table 1, A). Thus, addition of SRP does not confer a competitive
advantage to ppl86∆13-15 even though the signal sequence binds SRP. Therefore, we
conclude that a functional signal sequence is necessary for gaining an SRP-mediated
competitive advantage over nontranslating ribosomes.

46
Results

competitor competition
RNC
ribosomes -SRP +SRP

A ppl86 ∆13-15 (s) non-translating (s) yes yes

B ppl59 (s) non-translating (s) yes yes

C ppl59 (2) non-translating (1) yes yes

D ppl59 (1) non-translating (2) no no

Table 1: Binding of the signal sequence to the Sec61p complex is necessary for gaining a competitive advantage. (s)
indicates samples where ribosomes and RNCs were added to the microsomes at the same time. (1) & (2) indicate the
order in which ribosomes or RNCs were added to the microsomes.

In the ribosome competition assay, we also tested a shorter fragment of the wildtype
preprolactin chain containing the 59 amino terminal residues (ppl59). RNCs carrying ppl59
represent an early translocation intermediate; although the signal sequence can bind to SRP
(Jungnickel and Rapoport, 1995) it is too short to interact productively with the translocation
channel. Therefore, the RNCs bind to the ER membrane only loosely and the transition to
tight binding does not take place (Jungnickel and Rapoport, 1995). Since nascent chains of
membrane-bound RNCs bearing the ppl59 chain are accessible to proteinase K, we used a
sedimentation assay to measure RNC binding to microsomes. After incubation of the
membranes with ribosomes and RNCs, the sample was loaded on a sucrose cushion
containing physiological salt concentrations and the membranes were sedimented. The
sedimentation of the microsomal membranes was verified with immunoblots using antibodies
against Sec61α. The amount of membrane-bound ppl59 was determined by comparing the
pellet and supernatant fractions. In samples lacking microsomes no ppl59 was recovered in
the pellet fraction (data not shown). Competition experiments showed that RNCs carrying
ppl59 cannot gain an advantage over competing nontranslating ribosomes even in the
presence of SRP (table 1, B).

47
Results

Furthermore, when the membrane binding sites were saturated with nontranslating ribosomes
before addition of ppl59, the binding of the RNCs was prevented completely both in the
absence and presence of SRP (table 1, C). However, when RNCs carrying ppl59 were bound
to the microsomes first, they remained bound even when a large excess of nontranslating
ribosomes was added (table 1, D).
These data indicate that, besides SRP binding, a successful competition with nontranslating
ribosomes requires a productive interaction of the signal sequence with the Sec61p complex.
Also, in the case of ribosomes or RNCs that can only bind loosely to the Sec61p complex,
there is little exchange between the pools of free ribosomes and membrane-bound
ribosomes. It seems that a ribosome or RNC once bound to Sec61p remains bound and will
not be replaced by nontranslating ribosomes or by RNCs only capable of a loose interaction
with Sec61p. The situation is different for RNCs carrying ppl86 which in the presence of SRP
bind to microsomal membranes even when the membranes had been presaturated with
nontranslating ribosomes (see Figure 12).

3.2.6. SRP Binding Gives a Competitive Advantage to Ribosomes


Synthesizing Integral Membrane Proteins with A Signal Anchor

We wanted to verify that the SRP-mediated competitive advantage is granted not only to
ribosomes synthesizing secretory proteins, but also to those synthesizing a membrane
protein with a signal anchor. As a substrate we chose a short N-terminal fragment of the
integral membrane protein leader peptidase (Figure 17A). Leader peptidase has two
membrane anchors; the first membrane spanning domain serves as a signal anchor with the
N-terminus located in the lumen of the ER. A cytosolic loop and a second membrane anchor
follow, leaving the C-terminal portion of the protein in the ER lumen.

For competition experiments, ribosome-nascent chain complexes of a 57 residue N-terminal


fragment of leader peptidase were generated in the wheat germ system (lep57, Figure 17A).
Photocrosslinking verified binding of SRP to the signal anchor (data not shown). Membrane-
bound RNCs carrying lep57 reflect the high salt

48
Results

resistant stage of ribosome binding to the translocation channel; at this point the signal
anchor is bound by the Sec61p complex (W. Mothes, personal communication).

A leader peptidase

1 22 63 77 96 105

signal anchor membrane hydrophobic


anchor domain
lep57 57

∆63-77 ∆96-105

lepcyt 44 215

43 ∆63-77 ∆96-105
lepXa 215
Xa
cleavage site

B
Xa

Figure 17: Fragments of leader peptidase were used to study membrane binding of different populations of RNCs. (A)
The composition of wildtype leader peptidase is shown in comparison to other constructs used. lep57 represents a
fragment containing only the signal anchor and adjacent residues. Lepcyt is a fragment starting at position 44 and
lacking both the second membrane anchor and a short hydrophobic domain following the second anchor. lepXa
contains the first membrane anchor but both the second membrane spanning domain and the following hydrophobic
stretch are deleted. In addition, a
factor Xa cleavage site has been introduced at position 43. (B) Ribosomes synthesizing cytosolic domains of
membrane proteins remain bound to the translocation site. Using the lepXa construct shown above, it has been
shown that the translating ribosome remains bound to the translocation channel (Mothes et al., 1997) The cytosolic
domain of lepXa is found in close proximity to the Sec61p complex even after the preceding membrane anchor has
contacted the lipid phase of the ER membrane (left). This is the case even when the physical connection between
membrane spanning domain and cytosolic domain has been severed by treatment with factor Xa (right).

We used the sedimentation assay to monitor membrane targeting of RNCs carrying lep57 in
the absence and presence of competing nontranslating ribosomes. As for the secretory
protein preprolactin in the absence of SRP, nontranslating ribosomes were competing with
lep57 for membrane binding. Yet, when its signal anchor was bound by SRP, lep57 gained a
competitive advantage over nontranslating ribosomes (table

49
Results

2, A). Thus, we conclude that SRP binding gives a competitive advantage to RNCs carrying
either a signal sequence or a signal anchor.

3.2.7. Membrane Binding of Ribosomes Synthesizing Cytosolic Proteins

We also wanted to compare membrane binding of nontranslating ribosomes and of ribosomes


translating proteins destined to remain in the cytosol. As an example for a cytosolic protein,
we created a nascent chain of leader peptidase lacking all hydrophobic regions. It included
amino acids 44 to 215; both the second membrane anchor and the following short
hydrophobic stretch were deleted, leaving a total of 149 amino acids (Figure 17A, lepcyt).
First, we used ribosomes carrying the leader peptidase fragment for competition of
membrane binding of RNCs bearing the ppl86 chain. To this end, a sedimentation assay was
performed using ppl86 and lepcyt chains that had been radiolabeled with 35S-methionine.
Since the two populations of nascent chains were separated by SDS-PAGE, we were able to
monitor their membrane binding independently. When ppl86 and lepcyt RNCs were added to
the microsomes simultaneously in the absence of SRP, membrane binding of RNCs carrying
the ppl86 chain was reduced with addition of increasing amounts of RNCs carrying the lepcyt
polypeptide (table 2, B). Therefore we conclude that in the absence of SRP, RNCs
synthesizing cytosolic proteins can compete with RNCs carrying a signal sequence for
binding to the ER membrane. However, in the presence of SRP, ribosomes translating ppl86
were efficiently bound to the microsomes even when a large excess of RNCs carrying lepcyt
was present (table 2, B). This shows that SRP binding gives RNCs containing a signal
sequence a competitive advantage in membrane targeting over ribosomes synthesizing
cytosolic proteins.
After RNCs carrying ppl86 had been prebound to the membrane, even a large excess of
lepcyt RNCs did not replace them from the Sec61p complex (table 2, C). Conversely, when
ppl86 RNCs were added to microsomes where all binding sites had been saturated with
ribosomes carrying the lepcyt fragment, no membrane association of ppl86 was detected
without SRP. However, efficient targeting occurred in the presence of SRP (table 2, D).

50
Results

We also used RNCs carrying lepcyt in targeting assays with competing nontranslating
ribosomes. Both populations of ribosomes competed for binding sites at the ER membrane
(table 2, E). However, when microsomes had been saturated with one kind of ribosomes,
these were not replaced even after addition of a large excess of the other kind of ribosomes
(table 2, F and G). Next, we used an excess of ppl86 RNCs to compete ribosomes translating
lepcyt for binding to microsomes. For ppl86, efficient targeting took place whenever SRP was
present (table 2, H, I and J). Even when RNCs carrying lepcyt had been prebound to the
membrane, ppl86 RNCs with bound SRP were able to displace them (table 2, I)

Table 2: Competition experiments using RNCs carrying different fragments of leader peptidase. (s) indicates samples
where ribosomes and RNCs were added to the microsomes at the same time. (1) & (2) indicate the order in which
ribosomes or RNCs were added to the microsomes.

To summarize, ribosomes synthesizing cytosolic proteins as well as nontranslating ribosomes


compete for common binding sites with ribosomes translating secretory nascent chains or
membrane proteins. Binding of SRP confers a competitive advantage to RNCs that can
interact tightly with the Sec61p complex.

51
Results

In fact, these RNCs can secure a translocation site even when the ER membrane is saturated
with loosely bound ribosomes.

3.2.8. Ribosome Binding During Integration of Multispanning Membrane


Proteins

For integration of multispanning membrane proteins it has been suggested that the ribosome
experiences repeated targeting cycles after translation of cytosolic domains. However, recent
data indicate that the ribosome remains bound to the Sec61p complex while it is translating a
cytosolic domain.
These data were derived from experiments with translation intermediates of leader peptidase
containing only the signal anchor and the adjacent cytosolic domain. To allow detachment of
the cytosolic domain from the signal anchor, a consensus site for cleavage by factor Xa was
introduced at position 43 (Figure 17A, lepXa). After digestion with factor Xa, the ribosome-
associated fragment of lepXa is identical to lepcyt, the cytosolic fragment of leader peptidase
that has been used before (compare lepcyt and lepXa in Figure 17A).
Photocrosslinking experiments with membrane-bound ribosomes carrying the lepXa fragment
have shown that the cytosolic portion of lepXa remains in close proximity to the Sec61p
channel even after cleavage of the nascent chain by factor Xa (Mothes et al., 1997; see also
Figure 17B). Under these conditions, even though there is no physical connection between
cytosolic domain and membrane anchor, the ribosomes stay tightly bound to Sec61p (Mothes
et al., 1997).
We were interested in comparing the membrane binding of RNCs carrying the Xa-cleaved
lepXa to binding of ribosomes carrying the equivalent lepcyt chain. To this end, we performed
competition assays with nontranslating ribosomes and RNCs carrying ppl86.
First, ribosomes bearing the lepXa fragment were targeted to microsomes in the presence of
all cytosolic factors but SRP. Then, the sample was treated with factor Xa, resulting in
cleavage of about 50% of the lepXa chains. Next, competitor ribosomes were added, either
nontranslating ribosomes or RNCs carrying ppl86 with or without bound SRP. The efficiency
of membrane binding of the RNCs was tested with the sedimentation assay. Both the
uncleaved and the cleaved nascent chains as

52
Results

well as the ppl86 chain were detected when the sample was separated in SDS-PAGE,
allowing for independent monitoring of all populations of RNCs.

competitor competition
RNC
ribosomes -SRP +SRP

A lepXa (1) non-translating (2) no no

B lepXa (1) ppl86 (2) no no

Table 3: Competition experiments using membrane-bound ribosomes synthesizing cytosolic domains of integral
membrane proteins. (1)/(2) indicates that before addition of competing ribosomes, RNCs carrying lepXa were bound
to the ER membrane and digested with factor Xa.

As expected, nontranslating ribosomes and RNCs carrying ppl86 without SRP did not
compete off the Xa cleaved or the uncleaved lepXa RNCs (table 3, A and B). However, to our
surprise, RNCs carrying ppl86 were not able to displace ribosomes carrying the lepXa
fragments, even in the presence of SRP (table 3, B). The amount of cleaved and uncleaved
lepXa found in the membrane fraction was not reduced even when a 20fold excess of ppl86
RNCs was added, although ribosomes carrying the lepcyt chain were displaced by RNCs
carrying ppl86 with bound SRP (see table 2). Nevertheless, we did see a small increase of
ppl86 bound to microsomes in the presence of SRP, probably accounting for competition of
these RNCs with nontranslating ribosomes present in the sample. This observation confirms
that functional SRP was indeed bound to ppl86.
These results suggest that binding of ribosomes translating a cytosolic domain of a
multispanning membrane protein is distinct from binding of ribosomes synthesizing cytosolic
proteins. Our data indicate that a ribosome translating a multispanning membrane protein will
not leave the translocation site before integration of the membrane protein is completed.

53
Results

3.3. Structural Analysis of Ribosome Binding to the ER Membrane

Two different stages of ribosome binding to the translocation channel in the ER membrane
have been described (Wolin and Walter, 1993; Crowley et al., 1994; Jungnickel and
Rapoport, 1995). Nontranslating ribosomes, ribosomes synthesizing cytosolic proteins and
ribosomes carrying short secretory nascent chains bind only loosely to Sec61p. The
interaction is sensitive to high salt concentrations. A transition to tighter binding takes place
for ribosomes synthesizing secretory proteins upon elongation of the nascent chain and
binding of the signal sequence to the Sec61p complex. This tighter interaction is
characterized by resistance to high salt concentrations.
Biochemical data (Crowley et al., 1994) indicate that in the tight binding state the ribosome-
channel interaction might become much more intimate and that a continuous sealing exists
around the ribosome-membrane junction. Using single particle cryo electron microscopy, we
wanted to compare loose and tight ribosome binding to the translocon to determine whether
any differences between these stages can be detected on a structural basis. Furthermore, we
were interested in a comparison of the structural features of the purified Sec61p channel and
the native translocation channel, which has been shown to contain other integral membrane
proteins in addition to the Sec61p complex (Görlich et al., 1992a,b).

3.3.1. Preparation of Ribosome-Translocation Channel Complexes

We established a method for isolating stable complexes of ribosomes bound to translocation


channels in detergent solution. These complexes contained either nontranslating ribosomes
or RNCs carrying ppl86, representing loose or tight binding modes, respectively. For
determination of a 3D structure by single particle cryo electron microscopy it is essential to
generate a homogenous population of particles. Hence, we optimized the isolation procedure
to yield a maximum of ribosomes with an attached channel and to minimize loss of nascent
chains by hydrolysis of the peptidyl-tRNA.

54
Results

First, ribosome-nascent chain complexes of ppl86 were assembled in the rabbit reticulocyte
lysate system in the presence of reconstituted proteoliposomes containing purified Sec61p
and SRP receptor (Figure 18A, lane 1). It has been shown before that the 86mer of ppl
induces a high-salt resistant interaction of ribosome and translocation channel (Jungnickel
and Rapoport, 1995). By comparing the total number of ribosomes and the number of
nascent chains present in the in vitro translation reaction, we estimated that about 10-30% of
the ribosomes in the translation reaction carried a nascent chain (data not shown). After
binding of the RNCs, the proteoliposomes were floated in a sucrose step gradient under high
salt conditions. Membrane- associated RNCs were recovered in the floated fraction (Figure
18A, lane 5) whereas unbound RNCs and ribosomes bound only loosely to Sec61p remained
in the bottom part of the sucrose gradient (data not shown). The interaction of ribosomes and
membranes was mediated by the translocation channel since very little ppl86 was found in
the floated fraction when vesicles lacking Sec61p and SRP receptor were used (data not
shown). Taken together, these data indicate that after flotation only ribosomes tightly bound
to the Sec61p complex are recovered.

Following flotation, the membranes were solubilized in a buffer containing 1.5% digitonin.
Under these conditions, the Sec61p complex remains ribosome-associated whereas SRP
receptor is soluble. The ribosome-channel complexes were then pelleted to separate them
from an excess of unbound membrane proteins (Figure 18A, lane 9). Most of the 86mer
remained bound to ribosomes as peptidyl-tRNA, as demonstrated by precipitation with
cetyltrimethylammonium bromide (CTABr; Figure 18A, lane 10 vs. lane 9). In fact, the
complex of ribosome, 86mer and Sec61p channel proved to be very stable, even when
generated with native membranes instead of proteoliposomes (Figure 18B). Judged by
CTABr precipitation, the nascent chain stayed ribosome-associated for at least 5 hours
(Figure 18B, lane 14 vs. lane 13). In addition, the nascent chain remained protease-resistant,
indicating that it is located inside the ribosome-channel complex (Figure 18B, lane 15 vs. lane
13).
These data demonstrate that stable ribosome-channel complexes representing the tight
binding stage can be isolated.

55
Results

Figure 18: Preparation of ribosome-translocation channel complexes. (A) RNCs of ppl86 were assembled in
reticulocyte lysate in the presence of proteoliposomes containing purified Sec61p complex and SRP receptor. The
vesicles were subjected to flotation in a sucrose step gradient at either low (100mM potassium acetate) or high
(500mM potassium acetate) salt concentrations. Membranes with bound RNCs were solubilized in digitonin at the
indicated salt concentration and the ribosomes were isolated by centrifugation. Equivalent aliquots of the original
translation (T), after flotation (F100 and F500) or after sedimentation (P100 and P500) were analyzed by SDS-PAGE. Each
sample was also precipitated with CTABr (CTABr-ppt) and the amount of precipitable material is given below the
lanes (%). (B) As in (A), except that PKRM were used, and flotation and solubilization were performed at 500mM
potassium acetate. Aliquots of all samples were analyzed by CTABr precipitation (CTABr-ppt, lanes 2, 5, 8, 11 and
14) and in a protease-protection assay (proteinase K, lanes 3, 6, 9, 12 and 15). In addition, the final pellet fraction
was incubated on ice for either 1h or 5h to test the stability of the ribosome-channel complex (P1h, P5h). Four times
more material was loaded in the F and P samples than in T samples. (C) As in (A), except that a non-programmed
translation mix was used. The ribosomes in the original sample (T, 10% loaded) and in the final pellet fractions (P100
and P500) were detected by immunoblotting using an antibody raised against the ribosomal protein S26. ppl86 points
to the position of the nascent chain.

To prepare complexes of ribosomes loosely bound to the translocation channel we used


unprogrammed reticulocyte lysate as a source of nontranslating ribosomes. Flotation and
solubilization were carried out under low salt conditions. Immunoblotting with an antibody
directed against the ribosomal protein S26 demonstrated that nontranslating ribosomes were
present in the final fraction only at low salt concentration (Figure 18C, compare lanes 3 and
2). Again, this provided evidence that only ribosomes tightly associated to Sec61p are
recovered under high salt conditions.
Taken together, these data showed that we generated stable ribosome-translocation channel
complexes with or without a defined nascent chain. Isolation of the

56
Results

complexes allowed a structural analysis of the loose and tight stage of ribosome binding to
the Sec61p complex.

3.3.2. Structures of Ribosomes Bound to Purified Sec61p Complex

Previously, a 3D structure of nontranslating yeast ribosomes bound to purified yeast Sec61p


complex has been published (Beckmann et al., 1997). This structure, representing ribosomes
bound only loosely to the Sec61p complex, revealed a sizable gap between ribosome and
channel with just one connection bridging the ribosome-channel junction. To test whether the
ribosome-Sec61p channel junction becomes more intimate after binding of a signal sequence
to the Sec61p complex, we compared 3D maps of ribosomes bound to the channel formed by
the purified Sec61p complex in the absence and presence of a nascent chain.
Samples for electron microscopy were prepared as described above except that ppl86 was
translated without radioactive methionine. After sedimentation, the ribosome-channel
complexes were resuspended, frozen and analyzed by cryo electron microscopy. Ribosomes
were readily identifiable on the grid. In addition, some ring-like Sec61p complexes (Hanein et
al., 1996) were visible in the background. Using a previously determined 3D structure of the
rabbit ribosome as a first reference (Morgan et al., 2000) we determined 3D maps of the
isolated ribosome-channel complexes. Since the electron density is continuous, an
appropriate threshold level must be chosen to represent and interpret the structure. The
density above this threshold should correspond to the volume of the ribosome-channel
complex (see Materials and Methods). We have defined a ribosomal volume of 100% as the
threshold that encloses the calculated volume of a rabbit ribosome and, in addition, allows
expected features of the ribosome to be recognized. Due to some ambiguity in the volume
calculation, the appropriate threshold may deviate from this 100% value. Moreover, if some of
the ribosomes used for the structural analysis were lacking channels, a lower threshold may
be appropriate to visualize the full volume of the channel.

To our surprise, a comparison of the structures of ribosome-Sec61p channel complexes in


the absence (Figure 19, top row) or presence (Figure 19, bottom row)

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Results

of the ppl86 chain revealed no differences at a 25Å resolution (see Figure 19A, C, E and G
for frontal views of the ribosome-channel complexes, and Figure 19B, D, F and H for views
from the ER lumen; compare also table 4). Furthermore, the 3D maps are very similar to the
previously described structure of the yeast complex containing nontranslating ribosomes and
purified Sec61p complex (Beckmann et a., 1997; and our unpublished results).

Figure 19: Ribosomes and ribosome-nascent chain complexes associated with purified Sec61p complexes. (A) A 3D
map of ribosome-Sec61p channel complexes lacking a nascent chain is viewed along the plane of the ER membrane
(frontal view). The threshold level was chosen to encompass 100% of the ribosomal volume. The small (S) and large
(L) ribosomal subunits are indicated. (B) As in (A), but viewed from the ER lumen (bottom view). This view is
generated by a 90° rotation about the horizontal axis, followed by a 90° rotation in the image plane. (C) As in (A),
except that the threshold level was lowered to encompass 150% of the expected ribosomal volume. (D) As in (C), but
shown in bottom view. The Sec61p channel is shown as a circumference (outlined in gold) to reveal the ribosomal
nascent chain tunnel exit (TE). (E) A 3D map of ribosome-Sec61p channel complexes carrying the ppl86 chain is
shown in front view with the threshold level set to enclose 100% of the ribosomal volume. (F) Same as (E), but shown
in bottom view. (G) Same as (E), except with a threshold that encompasses 150% of the expected ribosomal volume.
(H) As in (G), but shown in bottom view. The Sec61p channel with an inserted nascent chain is shown as a
circumference (outlined in red) to reveal the ribosomal nascent chain tunnel exit.
The color code for the Sec61p complexes containing or lacking the ppl86 chain (-/+NC) is given as a vertical bar.
Scale bar=100Å.

At a 100% threshold level, the ring formed by the Sec61p channel had an outer diameter of
about 85Å and included a central pore (Figure 19B and F). The pore was aligned with the
nascent chain exit site of the large ribosomal subunit to provide a continuous passage from
the ribosomal peptidyltransferase center into the lumen of the endoplasmic reticulum (see
Figure 19D and H, the golden and pink channel outlines surround the tunnel exit sites,
indicated by TE). Between the ribosome and

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the Sec61p channel no connection was seen at the threshold level chosen here. Instead, as
described before for the yeast ribosome-channel complex (Beckmann et al., 1997; and our
unpublished results), a substantial gap of about 20Å width was present (Figure 19A and E).
The gap became narrower and some connections appeared when the threshold level was
lowered to enclose 150% of the calculated ribosomal volume (Figure 19C and G). However,
even in the presence of a nascent polypeptide chain engaged in translocation, the ribosome-
channel junction does not become more intimate and there were no significant differences
detectable between ribosome-Sec61p complexes containing and lacking a nascent chain.

Table 4: Summary of datasets

ribosomes membranes nascent chains particles resolution*

rabbit proteoliposomes none 9942 25Å

rabbit proteoliposomes ppl86 7902 25Å

rabbit PKRM none 6914 27Å

rabbit PKRM ppl86 6863 29Å

canine KRM mixed 6488 27Å


* Resolution was determined with the FSC 0.5 criterion. For each dataset, pairs of 3D volumes
were calculated with increasing numbers of particles up to the total number divided by 2.
The appropriate volumes were then compared and their resolution plotted as a function
of increasing particle number.
This allowed us to estimate the resolution of the final complete 3D dataset by extrapolation.

3.3.3. Ribosomes Bound to Native Translocation Channels

In addition to Sec61p, in native membranes other components of the translocation machinery


have been found to be tightly associated with ribosomes (Görlich et al., 1992a). These
membrane proteins might be part of the translocation channel. We therefore used PKRM to
pursue a structural analysis of complexes consisting of ribosomes bound to native
translocation channels.
Ribosome-channel complexes with or without ppl86 chains were prepared and subjected to
cryo electron microscopy as described above except that PKRM were added instead of
proteoliposomes containing purified membrane proteins. To remove

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Results

ER membrane proteins that are only loosely attached to the ribosome-channel complexes,
the membranes were always solubilized under high salt conditions.

The resulting 3D structures had a resolution of 27Å for ribosome-channel complexes without
a nascent chain and 29Å for complexes with a ppl86 chain (table 4). These 3D maps showed
essentially the same features as seen previously in structures of ribosomes bound to purified
Sec61p channel (Figure 20A, C, D and F, front view; Figure 20B and E, bottom view). As
before, there were only small differences between ribosome-channel complexes with or
without nascent chains. At a 100% threshold level (Figure 20A, B, D and E) no connection
was visible between ribosome and translocation channel. Yet, the central pore of the channel
was precisely aligned with the nascent chain exit site of the large ribosomal subunit. Again, a
20Å gap was present between ribosome and channel. When the threshold level was set to
150% of the ribosome volume, the gap became narrower and a few connections appeared
(Figure 20C and F).
Interestingly, in contrast to the channel formed by the purified Sec61p complex, an additional
domain was visible on the lumenal side of the native translocation channel. This domain was
precisely oriented with respect to the ribosome and the channel (to be discussed in more
detail later).

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Results

3.3.4. Ribosome-Channel Complexes with a Mixed Population of Nascent


Chains

Using ppl86 as a model for a nascent chain that is inserted into the translocation channel, we
have not been able to detect any changes in the ribosome-channel junction during the
transition from loose to tight ribosome binding to the Sec61p channel. To exclude the
possibility that our results were biased by the choice of ppl86 as the nascent chain we wished
to examine ribosome-channel complexes representing different stages of translocation of a
wide range of substrates. We therefore prepared ribosome-channel complexes derived from
salt-washed microsomes (KRM). Ribosomes bound to these native ER membranes carry a
mixture of endogenous nascent chains engaged in translocation, presumably nascent chains
of secretory and membrane proteins at later stages of translocation. By using ribosome-
channel complexes prepared from KRM we also compared endogenously formed channels to
translocation complexes assembled in vitro.
After treating canine rough microsomes with high salt concentrations to remove ribosomes
without nascent chains, these KRM were solubilized in digitonin under high salt conditions.
Then, the ribosome-channel complexes were pelleted and analyzed by cryo electron
microscopy as before.
A 3D map of canine ribosome-nascent chain complexes bound to canine Sec61p channel
was generated at 27Å resolution (table 4). The canine ribosome proved to be very similar to
the rabbit ribosome. Also, all features described previously for structures of ribosome-channel
complexes were visible in the map derived from KRM (Figure 21A to C). The ribosomal exit
site was found aligned with the central pore of the Sec61p channel (Figure 21C, the pink line
shows the contour of the channel, TE marks the tunnel exit). As before in structures derived
from PKRM, the prominent lumenal domain was visible (Figure 21A and B). Even with
complexes containing a mixed population of nascent chains, no connections were seen
between ribosome and channel and a gap of about 20Å was present at a threshold level of
100% (Figure 21A).

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Results

3.3.5. The Ribosome-Channel Junction

Data derived from biochemical studies have indicated that the ribosome-membrane junction
becomes much more intimate when a nascent chain is inserted into the translocation channel
(Crowley et al., 1994). However, our structural data show that at least at the resolution
presented here, no significant differences are seen between ribosome-channel complexes
lacking nascent chains and those actively engaged in translocation. Therefore, we wished to
study the ribosome-channel junction in more detail. To do so, we chose the structure of
ribosome-channel complexes derived from KRM as an example.
As for all structures described here, at a 100% threshold level, no connection was visible
between ribosome and Sec61p channel (Figure 21A). Instead, a sizable gap of about 20Å
was present. When the threshold level was lowered to include 150% of the calculated
ribosome volume, the gap narrowed and a few connections between channel and large
ribosomal subunit appeared (Figure 22A). At a threshold level set to 200%, the links
connecting ribosome and channel became more prominent (Figure 22B). However, the
ribosome-channel interface was not completely filled with electron-dense material even
though at 200% the threshold level is so low that some background noise started to appear
(shown in gray). In Figure 23A, a slice through the same ribosome-channel complexes is
shown, contoured to represent threshold levels set to 100% (green), 150% (yellow) and 200%
(white). The plane of the membrane (M) is indicated by white lines, PTC indicates the position
of the peptidyltransferase center in the ribosome. Even at a contour level of 200%, a
continuous passage from the ribosomal nascent chain tunnel into the cytosol was revealed
(Figure 23A, dotted line). Clearly, a 200% threshold level is too low, since both the channel
pore and an uninterrupted ribosomal tunnel from the PTC to the nascent chain exit site are no
more visible (Figure 23A, white contour line). Since the gap is still visible at this unreasonably
low threshold level, we conclude that it is a genuine feature of the ribosome-channel junction.

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Results

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Results

A statistical analysis further strengthened this conclusion. We identified regions that


contained neither protein nor RNA (Figure 23B; the blue areas are significant at a confidence
level greater than 99%, T indicates the tunnel within the ribosome). These data show that the
peptidyltransferase center, the central pore of the translocation channel and the gap between
ribosome and channel are equally empty. A similar analysis performed with all other 3D
structures confirmed that the gap is a significant feature of the ribosome-membrane junction.

We then compared the position of links between the large ribosomal subunit and the Sec61p
channel in all structures presented here and in a similar structure of yeast nontranslating
ribosomes bound to yeast Sec61p complex.
In Figure 24, an overlay is shown of slices through ribosome-channel complexes at the level
of the ribosome-channel junction. The complex derived from S. cerevisiae is indicated by the
dark green contour line of the ribosome. Mammalian ribosome-channel complexes containing
either purified Sec61p channel or native channels, both in the absence and presence of a
nascent chain are represented by all other contour lines. The light green channel outline
represents the channel map derived from PKRM without nascent chains. The slices were
aligned using the ribosomal tunnel exit as a reference (indicated by the arrow).
Three connections (dark shaded areas inside the channel contour) were identified as present
in all of the structures, another connection (lighter shaded area above the tunnel exit) was
present in most of the structures. Since the slices are superimposed onto each other, the
darkest areas represent mass found most frequently in one location, whereas lighter areas
indicate electron dense mass found only in some maps.

The conserved links between ribosome and channel are arranged around the central pore of
the channel in a horseshoe shape. In structures derived from native ER membranes, the
opening of the horseshoe towards the cytosol points away from the lumenal domain. There
were never any connections visible on the side of the channel where the lumenal domain
emerges nor in any position to completely seal the ring around the central pore (compare to
Figure 22) indicating that the location of the gap in the ribosome-channel junction is
conserved among all structures analyzed.

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Results

Figure 24: A discrete number of connections between ribosome and translocation channel are present in conserved
positions. An overlay is shown of sections through ribosome-channel complexes in the plane of the ribosome-channel
junction. The ribosome-channel complexes contained either nontranslating yeast ribosomes bound to Sec61p
complex purified from S. cerevisiae (contoured in dark green) or mammalian ribosomes bound to mammalian
translocation channels derived from native membranes or from reconstituted proteoliposomes containing purified
Sec61p complex (all other contours). The maps are aligned with respect to the ribosomal nascent chain tunnel exit
(indicated by the arrow). The outline of the translocation channel derived from PKRM in the absence of a nascent
chain is given as a reference (light green outline). Scale bar=100%.

We then raised the threshold level for contouring of the ribosome-channel complexes to
include only high-density structures representing the ribosomal RNA rather than ribosomal
proteins. In these maps distinct features of the ribosome were found to be located at positions
were the conserved links to the channel had been seen (data not shown) suggesting that
specific regions of ribosomal RNA take part in establishing the connection to the translocation
channel.

Taken together, these data show that the ribosome is bound to the Sec61p channel by a
discrete number of links, presumably connected to or even formed by distinct regions of
ribosomal 28S RNA. These links are precisely located with respect to both the ribosome and
the translocation channel. In addition, our results indicate that even when a nascent chain is
being transferred through the channel, the ribosome-membrane junction is not completely
sealed towards the cytosol.

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Results

3.3.6. Comparison of Purified Sec61p Channel and Native Translocation


Channel

While our data show that the ribosome-channel junction is similar for structures derived from
the purified Sec61p complex or from native membranes, the channels themselves differ
significantly. In Figure 25, the purified Sec61p channel without a nascent chain (Figure 25A,
left panel, and Figure 25B) and the native translocation channel (derived from KRM, Figure
25A, middle panel, and Figure 25C) are depicted at a threshold level of 110%. The channels
are shown without the ribosomes either as viewed from the lumen of the ER (Figure 25A, top
row), as viewed from the ribosome (Figure 25B and C, left panel) or in frontal view (Figure
25A, bottom row, and Figure 25B and C, middle and right panel).

Compared to the purified Sec61p channel, the native channel contained an additional lumenal
domain (Figure 25A, middle vs. left panel) that was precisely oriented with respect to both
ribosome and membrane-spanning regions of the channel (see also Figure 21A and B). Part
of the lumenal domain was found to be positioned over the central pore of the channel (Figure
25A, middle panel).
In addition, channels derived from native membranes appeared to be elliptical and larger;
125Å in the longest dimension compared to 85Å of the purified Sec61p channel.
When purified and native channels were overlaid (Figure 25A, right panel) it became clear
that the region without the lumenal domain seemed to be similar. The most striking
differences were visible where the lumenal domain emerged from the channel. This part of
the channel wall is extended and the central pore is enlarged (Figure 25A, top row, right). The
increased pore size (25x50Å at a 100% threshold) is consistent with results previously
described using freeze-fracture electron microscopy (Hanein et al., 1996).

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Results

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Results

To study the actual pore in more detail, we compared purified and native translocation
channels after sectioning them along the axes marked (a) and (b) in Figure 25A. The Sec61p
channel derived from proteoliposomes containing purified membrane proteins has a cup-
shape with a larger opening towards the ribosome (about 50Å diameter) and a more narrow
opening (about 25Å) towards the lumen of the ER (Figure 25B, middle and right panel). The
channel derived from native membranes looked similar when sectioned along the short axis
(Figure 25C, middle panel, a in Figure 25A). However, a section cut 60° away (b in Figure
25A) revealed a much larger opening throughout the channel (Figure 25C, right panel).

3.3.7. The Nature of the Lumenal Domain

One of the most interesting features seen in structures derived from the native ER
membranes is the additional lumenal domain. The position of the lumenal domain with
respect to the central pore of the translocation channel seems to allow contact with the
nascent chain when it is emerging from the channel. Therefore, the lumenal domain might be
actively involved in protein translocation. We wished to identify the component of the
translocation channel that forms the lumenal domain. Thus, we analyzed the protein
composition of ribosome-channel complexes prepared from KRM. To release non-ribosomal
proteins associated with the ribosome-channel complexes, we performed an extraction with
very high salt concentrations (1200mM potassium acetate) and puromycin (to release the
nascent chains). We assumed that all proteins that remain ribosome-associated under the
conditions we used for isolating the ribosome-channel complexes (500mM potassium acetate,
1.5% digitonin) would most likely be integral membrane proteins. Thus, to enrich hydrophobic
proteins, the sample was extracted with Triton X-114 before separation by SDS-PAGE.
Staining with Coomassie Blue revealed that only two other complexes of membrane proteins
are present at a concentration similar to the one of the Sec61p complex (Figure 26, lane 2).

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Results

Figure 26: Ribosome-associated membrane proteins present in ribosome-channel complexes derived from KRM.
Ribosome-channel complexes derived from KRM were treated with 1200mM potassium acetate and puromycin to
release the nascent chains and extract ribosome-associated proteins. After the high salt/ puromycin treatment, the
ribosomes were pelleted and the supernatant was extracted with Triton X-114 to enrich hydrophobic proteins. The
samples were analyzed by SDS-PAGE and Coomassie staining. For comparison, an aliquot of KRM is shown in lane
1. Lane 2 shows proteins that have been extracted from ribosome-channel complexes by the treatment described
above. In addition to the Sec61p complex (Sec61α and β are indicated), three subunits of oligosaccharyl transferase
(indicated by OST) have been found and the four subunits of the TRAP complex (marked by TRAPα, β, γ, δ).

One protein complex was identified as oligosaccharyl transferase (OST; Kelleher et al., 1992)
which attaches carbohydrate chains to newly translocated nascent chains. Three proteins of
the OST were present in the ribosome-channel complexes: P48 and ribophorin I and II. The
other membrane proteins were identified as the four subunits of the translocon associated
protein complex (TRAP; Wiedmann et al., 1987; Görlich et al., 1990).

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Discussion

4. DISCUSSION

In this thesis project, cotranslational ER targeting of secretory proteins and binding of


ribosomes to the ER membrane was studied. First, calmodulin was described as a novel
cytosolic interaction partner for signal sequences. Next, ribosome binding to the membrane of
the endoplasmic reticulum was studied, resulting in a new view of how differences in binding
result in specificity of translocation. Last, a structural analysis of ribosome-translocation
channel complexes with and without a nascent chain gave new insights in the nature of the
ribosome-membrane junction and the anatomy of the channel.

4.1. Interaction of Calmodulin with Signal Sequences

We have found that the cytosolic protein calmodulin binds to signal sequences of nascent
polypeptide chains in a Ca2+ dependent manner. The interaction is sensitive to inhibitors of
calmodulin function. When SRP or microsomes are present, calmodulin binding to signal
sequences is reduced.

4.1.1. Substrate Binding by Calmodulin

Calmodulin is a small cytosolic protein, known to be a key-player in the modulation of Ca2+


dependent signal transduction pathways by regulating the activity of many different enzymes,
such as calcineurin, calmodulin kinase I and II or elongation factor 2 kinase (for review see
James et al., 1995).
Based on the crystal structure of calmodulin (Babu et al., 1985; Babu et al., 1988) and on
data derived from photo-labeling studies (Kauer et al., 1986; O’Neil et al., 1989; O’Neil and
DeGrado, 1989) it is well understood how calmodulin binds its substrates (reviewed in O’Neil
and DeGrado, 1990). Upon Ca2+ association with calmodulin, a conformational change
creates a peptide binding site by bringing two hydrophobic patches closer together which are
located in the globular domains at the N- and C-termini. The extended α-helix connecting the
N- and C-terminal lobes could serve as a flexible tether to adjust the peptide binding domain

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Discussion

to a wide range of substrates with a high sequence variability. A common feature of most
calmodulin binding motifs is a peptide adopting an amphiphilic α-helical structure (so-called
baa-peptides, for basic, amphiphilic α-helices). It has been suggested that signal sequences
can adopt α-helical structures (Gierasch, 1989; McKnight et al., 1989; Plath et al., 1998),
maybe explaining how calmodulin can recognize signal sequences as a substrate.
Using model peptides carrying a photoaffinity label it has been shown that two methionine
residues of the C-terminal lobe and another methionine residue at the N-terminal lobe are
contacting hydrophobic residues of bound peptides (O Neil et al., 1989). For SRP, similar to
calmodulin, it has been suggested that methionine residues are involved in recognition of
substrates (Bernstein et al., 1989). The methionine-rich M-domain of the 54kDa subunit of
SRP (SRP54) contains the signal sequence binding site (Zopf et al., 1990; Lütcke et al.,
1992). In fact, the crystal structure of Ffh, the prokaryotic homologue of SRP54 has revealed
that the M-domain forms a deep hydrophobic groove lined with highly flexible methionine side
chains (Keenan et al., 1998. Given that both calmodulin and SRP seem to use the same
principle for association with potential substrates, it is not too surprising that nascent signal
sequences can interact with calmodulin. However, it is unlikely that SRP will bind to targets of
calmodulin since a stable association of SRP with signal sequences requires the presence of
a ribosome.

There is also evidence that the hydrophobic patches at N- and C-termini of calmodulin
interact simultaneously with opposite ends of the peptide (O’Neil and DeGrado, 1989). Using
a site-specific photocrosslinking approach, we have shown that crosslinks to calmodulin from
different positions in the signal sequence each have a distinct mobility when separated by
SDS-PAGE. These different mobilities frequently result from crosslinks to different domains of
the crosslinking partner. In fact, in S. cerevisiae it has been shown that crosslinks of different
mobility from the signal sequence of a secretory protein to the α-subunit of the Sec61p
complex correspond to crosslinks to either one of two different membrane anchors of Sec61α
(Plath et al., 1998). They occur with a periodicity of three to four amino acids, suggesting that
the signal sequence is in an α-helical conformation that contacts one domain of Sec61α on
one side and another domain of Sec61α on the other side. Strikingly, our

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Discussion

preliminary data indicate that a similar crosslinking pattern can be observed in


photocrosslinking experiments using signal sequences bound to either SRP or calmodulin.
This could suggest a general mechanism for signal sequence recognition in which at least
two regions of the binding partner must contact appropriate regions of the substrate to allow a
stable interaction. Experiments using modified calmodulin and SRP with an internal cleavage
site for a site-specific protease, for example factor Xa, could allow mapping of domains
interacting with signal sequences and would subsequently lead to a more thorough
understanding of the mechanism of signal sequence binding and recognition.

4.1.2. Protein Translocation And Calmodulin

Binding of signal sequences by calmodulin has only been observed in in vitro experiments. It
would be interesting to know whether this interaction can also occur in vivo and whether
calmodulin is involved in the cotranslational translocation pathway. We have shown that in
vitro signal sequences are preferentially bound by SRP when both SRP and calmodulin are
present, suggesting that SRP has a higher affinity for signal sequences. However, in the cell
there is at least a 1000 fold excess of calmodulin over SRP (10-100µM versus 10nM;
Martoglio et al., 1997; Siegel and Walter, 1988) indicating that in spite of the lower affinity
calmodulin may bind to signal sequences in vivo. Our data also show that calmodulin can
easily dissociate from signal sequences and that the interaction of calmodulin and signal
sequences would not interfere with translocation of nascent chains across the ER membrane.

In S. cerevisiae, CMD1, the gene encoding the calmodulin homologue, is essential (Davis et
al., 1986). Using a yeast strain with a temperature-sensitive allele of CMD1, it has been
shown that the biogenesis of the vacuolar protein carboxypeptidase Y and of the secretory
protein invertase is not dependent on the function of calmodulin (Kübler et al., 1994). Yet, for
invertase we have demonstrated that calmodulin can interact with the nascent polypeptide
chain in vitro. Assuming that the primary cytosolic interaction partner of signal sequences in
mammalian cells is SRP, it is conceivable that binding of signal sequences by calmodulin
does not play a role under normal conditions but that it might become important under certain
cell stress

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Discussion

conditions. In the event of an increased expression of proteins destined to cross or to be


integrated into the membrane of the endoplasmic reticulum, the amount of available SRP
might not be sufficient to mediate targeting. In this case, calmodulin might bind to signal
sequences.

In the cell, calmodulin is involved in modulation of Ca2+ dependent signal transduction


pathways, raising the question of whether the interaction of signal sequences with calmodulin
could affect any of these pathways. If we assume that calmodulin can function as a backup
binding partner for signal sequences in the case of an overload of signal sequence-bearing
nascent chains, this interaction could either trigger inhibition of protein translation or
stimulation of the expression of proteins involved in targeting or ER translocation.
In fact, for the unfolded protein response in S. cerevisiae it has been shown before that
modulation of translation can result from an overload of secretory proteins (for review see
Sidrauski et al., 1998).

Supporting our finding that signal sequences are substrates for calmodulin, another study
demonstrated that calmodulin can interact with fragments of signal sequences that have been
cleaved off nascent chains during translocation across the ER membrane and that
subsequently have been released into the cytosol (Martoglio et al., 1997). Again, the in vivo
function of this interaction remains elusive. Martoglio et al. speculated that fragments of a few
specific signal sequences are released into the cytosol to function as calmodulin antagonists.
However, based on our results we consider it likely that binding to calmodulin is an intrinsic
property of most if not all signal sequences. Clearly, more work needs to be done to
understand if and how the interaction of calmodulin with signal sequences is important in
secretion of proteins from the cell.

4.2. Regulation of Ribosome Binding to the ER Membrane

We have found that SRP-independent targeting of ribosome-nascent chain complexes to the


membrane of the ER occurs in the presence of all cytosolic factors, including the nascent
polypeptide-associated complex (NAC). Furthermore, we have

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Discussion

demonstrated that in a system containing complete cytosol but lacking SRP, all ribosomes
compete for the same membrane binding sites, independent of whether or not they carry a
nascent chain with a signal sequence. In the presence of SRP, RNCs with a signal sequence
or a signal anchor have a competitive advantage over other ribosomes. However, binding of
SRP to the signal sequence is not sufficient for gaining the advantage; in addition, the
nascent chain has to insert productively into the translocation channel.
Experiments using reconstituted proteoliposomes containing purified components of the
translocation machinery have shown that the Sec61p complex and the SRP receptor are
necessary and sufficient for conferring the competitive advantage to RNCs with bound SRP.
We have also found that all ribosomes that can interact only loosely with the Sec61p complex
behave identical with respect to binding to the ER membrane. It does not matter whether
these are nontranslating ribosomes, RNCs of cytosolic proteins or ribosomes carrying a
nascent chain with a signal sequence that is not yet long enough to be recognized by the
translocation channel.
Our data also suggest that in the absence of SRP there is little exchange between the pools
of free ribosomes and ribosomes bound to the ER membrane. In the presence of SRP,
however, RNCs carrying a signal sequence with bound SRP can displace loosely bound
ribosomes. In contrast, ribosomes translating cytosolic domains of membrane proteins cannot
be competed off by these RNCs.

4.2.1. SRP-Independent Targeting in the Presence of Cytosol

Based on experiments employing salt-washed RNCs, NAC has been suggested as a general
cytosolic inhibitor of ribosome binding in the absence of SRP (Wiedmann et al., 1994; Lauring
et al., 1995a). Addition of SRP would allow RNCs with a signal sequence to overcome the
inhibitory effect of NAC (Lauring et al., 1995b).
Our results do not support a model in which specificity of targeting is achieved by NAC-
mediated inhibition of ribosome binding. We rather suggest that SRP functions as a positive
effector to allow efficient targeting of RNCs carrying a signal sequence, in spite of non-
selective binding of ribosomes to the ER membrane. Although we cannot explain the
divergence of the results we believe that experiments in a

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Discussion

complete translation system may be more meaningful than experiments using salt-washed
RNCs, as they should more closely reflect the physiological conditions.

We have demonstrated SRP-independent targeting in two translation systems. However,


based on early studies, targeting was always believed to be dependent on SRP (Walter and
Blobel, 1980; Walter et al., 1981). This paradox can be explained by three major differences
in the experimental settings- all three resulting in a higher efficiency of RNC binding to the ER
membrane in the absence of SRP.
The first difference is the use of PKRM in our system. PKRM are microsomes that have been
stripped of endogenously bound ribosomes and RNCs. They offer a higher concentration of
unoccupied ribosome binding sites compared to previously used microsomes covered with
either endogenous ribosomes (RM, KRM) or ribosome remnants (EKRM). A second
difference might be the enhanced efficiency of current translation systems. An increased ratio
of RNCs to total ribosomes would result in an increased number of RNCs bound to the ER
membrane even when all ribosomes compete for the same binding sites. The last difference
is uncoupling of targeting from translation. In our experiments truncated nascent chains of
optimal length for productive membrane interaction were used, as a result creating a large
time window for insertion into the membrane. Also, when targeting is initiated after translation
has been stopped, a higher concentration of microsomal membranes can be added since
their inhibitory effects on translation can be neglected.

Based on these assumptions we have to ask how significant SRP-independent targeting is in


the context of a cell. There, translation and translocation would occur simultaneously. Hence,
SRP-independent targeting would be reduced to a small time window before elongation of the
nascent chain, since folding of the polypeptide would result in a conformation in which the
signal sequence is no longer available for a productive interaction with the Sec61p complex.
Nevertheless, the fact that we have found no evidence for a cytosolic inhibitor of ribosome-
membrane interaction suggests that all ribosomes can bind to the ER membrane in vivo. This
hypothesis is supported by the observation that a sizable population of ribosomes is removed
from rough microsomes under high salt conditions (Kalies et al., 1994). Our data indicate

76
Discussion

that RNCs with a signal sequence and bound SRP could simply displace these ribosomes,
consequently securing a channel for translocation of the nascent chain.

4.2.2. How Does an SRP-Advantage Work?

Experiments using reconstituted proteoliposomes containing purified membrane proteins


showed that the interaction of SRP with its receptor is necessary to allow preferred binding of
RNCs to the Sec61p complex. But how does the SRP-SRP receptor interaction confer a
competitive advantage to these RNCs?

A simple explanation would be that binding of SRP to SR provides an additional link between
the RNC and the membrane. We have shown that mere binding of SRP to RNCs is not
sufficient for getting an advantage over competing ribosomes. RNCs of the mutant ppl86∆13-
15 chain do not gain a SRP advantage although photocrosslinking experiments have shown
that the mutant signal sequence still binds SRP with an efficiency of 70% compared to
wildtype ppl86 (Jungnickel and Rapoport, 1995). Certainly, the affinity of the mutant signal
sequence for SRP could be lower than the crosslinking data suggest. However, likewise no
SRP advantage is given to RNCs carrying ppl59, nascent chains that interact well with SRP
but are too short to insert into the translocation channel (Jungnickel and Rapoport, 1995).

One could speculate that binding of SRP to its membrane receptor grants a kinetic
advantage, thus creating a larger time window for a productive interaction of the nascent
chain and the Sec61p channel. Once the signal sequence would be recognized by Sec61p
and inserted into the channel, the ribosome would remain tightly bound to the membrane.
This could explain why in a case where the signal sequence cannot insert properly, such as
for ppl59 or ppl86∆13-15, the kinetic advantage provided by the SRP-SR interaction would
not be sufficient to allow successful competition. In contrast, in an in vivo situation the time
window might be large enough so that RNCs whose nascent chain is initially too short for
binding of the signal sequence by the Sec61p complex, may elongate the nascent chain and
insert it productively into the translocation channel.

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Discussion

The model described above is based on the assumption that at the ER membrane rapid
association and dissociation of ribosomes take place. Both on and off rate of ribosome
binding to the Sec61p complex would be high. In contrast, our data show that the exchange
between the pools of free ribosomes and those bound to the ER membrane is rather slow.
Since ribosome association to the ER membrane is a fast process, slow dissociation is
presumably responsible for the low exchange rate. Provided that indeed the off-rate of
ribosome binding to the ER membrane is low compared to the on-rate, our data indicate that
the SRP advantage might be based on a more complex mechanism. We have shown that
RNCs of ppl86 with bound SRP can displace nontranslating and other loosely bound
ribosomes from the ER membrane. The fact that in the presence of SRP the displacement
occurs so rapidly could indicate that it is an active process. The only membrane components
necessary for gaining an SRP-advantage are the SRP receptor and the Sec61p complex.
Therefore they might cooperate in a so far unknown manner to actively displace other
ribosomes to secure a translocation site for RNCs with a signal sequence and bound SRP.
Our data derived from experiments with ppl59 and ppl86∆13-15 also show that the SRP
advantage does not originate from the initial interaction of RNCs with bound SRP and
membrane but that it is implemented at a later point. This point may be the assembly or the
gating of the translocation channel.
The Sec61p complex is thought to form the translocation channel by assembling into trimeric
or tetrameric structures (Hanein et al., 1996) but otherwise little is known about the dynamics
of translocon formation. One could envision that to create a new translocon the SRP receptor
can recruit subunits from a multimeric channel even if this channel has a ribosome bound to
it. The insertion of the nascent chain into the translocation channel would stabilize the newly
formed structure. Our data show that ribosomes synthesizing cytosolic domains of membrane
proteins cannot be displaced from their translocation sites. This is true even when the
physical connection between ribosome and preceding membrane anchor is disrupted by
cleavage with a site-specific protease, suggesting that under these conditions the translocon
remains stable. If SRP receptor plays an active role in recruiting subunits for a new translocon
it will most likely not be able to do so from stabilized channels.

78
Discussion

In summary, we suggest that SRP and SRP receptor could actively assist in the recruitment
of a translocation site for RNCs with a signal sequence. As has been proposed before,
insertion of the signal sequence into the translocation channel would alter the channel and/or
the ribosome-channel junction. The new arrangement would not depend anymore on the
presence of a nascent chain in the channel and therefore comprise more than just a link
between ribosome and channel provided by the nascent chain.
Based on these results, one could have expected significant changes in the ribosome-bound
translocation channel after insertion of the nascent chain. However, at a resolution of 25-27Å,
a structural analysis of ribosome-channel complexes with and without an inserted nascent
chain (see below) did not reveal major differences. Further experiments exploring the function
of SRP receptor and the dynamics of the translocon in conjunction with a high resolution 3D
structure of the ribosome-channel complex will probably allow a better understanding of
membrane binding of RNCs with bound SRP compared to binding of other ribosomes.

4.2.3. Other Aspects of Unspecific Ribosome Binding to the ER

As mentioned before, the fact that we failed to detect an inhibitory effect of cytosolic factors
on binding of ribosomes to the membrane of the ER probably means that most translocation
sites on the ER surface are occupied at all times. Again, this would not hinder efficient
translocation of secretory proteins across the ER membrane since SRP binding allows RNCs
with a signal sequence to displace most other ribosomes from the membrane.
Therefore, both nontranslating ribosomes and ribosomes synthesizing a variety of proteins
could become associated with the membrane of the endoplasmic reticulum. In fact,
translation could start at the ER membrane. It has been shown that even nascent chains
ultimately destined for the cytosolic compartment can be translated by ribosomes bound to
the ER membrane. These nascent polypeptide chains would most likely not enter the lumen
of the ER since the Sec61p complex executes a second signal sequence recognition step
(Jungnickel and Rapoport, 1995). Therefore, polypeptides without a functional signal
sequence are excluded from translocation across the ER membrane.

79
Discussion

Little is known about what is happening to a translocon after translation and subsequently
translocation has been terminated. The ribosome may remain bound to the membrane until it
is displaced by a RNC with bound SRP. Alternatively, it could dissociate from the
translocation channel after termination of translocation. Similarly, the translocon with all
associated factors could disassemble with the components returning to a pool of free subunits
or it could stay in the assembled state for another round of translocation.
In freeze-fracture images of PKRM, ring-like structures of the Sec61p complex were detected
even though in these microsomes the majority of ribosomes had been removed by treatment
with puromycin and high salt (Hanein et al., 1996). These data suggest that even after
dissociation of the ribosome the translocation channel remains intact, although the release of
the nascent chain by puromycin may not reflect physiological termination of translation.

4.2.4. Consequences for Integration of Multispanning Membrane Proteins

So far, two models existed for how the next transmembrane domain is integrated into the
membrane after a cytosolic loop has been synthesized. According to the first model, the
ribosome leaves the translocation site to translate the cytosolic domain (Blobel, 1980;
Sabatini et al., 1982; Kuroiwa et al., 1996). The following membrane anchor is bound by SRP
when it emerges from the ribosome. Subsequently, the RNC is retargeted to the same or a
different Sec61p channel. However, so far there is no evidence that the integration of
multispanning membrane proteins depends on SRP other than for the initial targeting step.
The second model assumes that the ribosome never leaves the translocation site and that the
cytosolic domain is synthesized by a membrane bound ribosome (Wessels and Spiess, 1988;
Mothes et al., 1997). This model is supported by our finding that ribosomes translating a
cytosolic domain of a multispanning membrane protein will not be displaced by any other
ribosomes, even if they carry a nascent chain with a signal sequence and bound SRP.

80
Discussion

4.3. 3D Structures of Ribosome-Translocation Channel Complexes

We have performed a structural analysis of loose and tight stages of ribosome binding to the
membrane of the endoplasmic reticulum using single particle cryo electron microscopy. In this
study, 3D structures of ribosome-channel complexes in detergent solution were generated in
the presence or absence of nascent chains. The translocation channels were either derived
from vesicles containing purified Sec61p complex or from native microsomes.

Several common features were observed in all 3D structures examined. The translocation
channel is assembled into a disc containing a central pore and precisely positioned with
respect to the ribosome. As a result, the channel pore is aligned with the exit site of the
ribosomal nascent chain tunnel, providing a continuous passage for the nascent polypeptide
from the peptidyltransferase center into the lumen of the ER. A discrete number of
connections link the large ribosomal subunit to the translocation channel, bridging a sizable
gap between ribosome and channel.
To our surprise, no significant differences were seen between ribosome-channel complexes
carrying or lacking a nascent chain at a resolution of 25-29Å. Instead, we found that channels
derived from native microsomes or from vesicles containing purified Sec61p complex differ
remarkably.

4.3.1. The Ribosome-Membrane Junction

In all ribosome-channel complexes analyzed, a gap of about 20Å was seen between the
ribosome and the translocation channel. This result is somewhat surprising. Based on earlier
biochemical studies, a continuous seal had been predicted for the ribosome-membrane
junction, particularly in the presence of a membrane-inserted nascent chain when gating of
the translocation channel was thought to induce a much more intimate ribosome-channel
junction (Crowley et al., 1994). In contrast, we show that even with a nascent chain the
connection between ribosome and channel is established by a discrete number of links
conserved between mammals and yeast. These links are arranged horseshoe-like around the
pore of the channel with the opening of the horseshoe providing a passage to the cytosol.

81
Discussion

On the ribosomal side, the links are connected to distinct high density features, most likely
representing RNA. This is in good agreement with recently published data, showing that the
28S RNA of the large ribosomal subunit mediates ribosome binding to the membrane of the
ER (Prinz et al., 2000). Specific RNA sequences are involved, supporting the idea that a
limited number of connections in conserved positions exist. It seems possible that the links
between ribosome and translocation channel that are visible in the 3D maps are actually
formed by ribosomal RNA rather than proteins.

For several reasons we consider it unlikely that the gap between ribosome and translocation
channel is an artifact. First, it has been observed in two independent studies, with different
sources of material as well as different methods of complex preparation and analysis
(Beckmann et al., 1997 and this thesis). Second, the gap is present in 3D maps calculated
either with compensation of the contrast transfer function (Beckmann et al., 1997) or by
restricting the resolution, such that the contrast transfer function does not play a critical role
(this study). Third, the gap is still seen at unrealistically low threshold levels. Fourth, a
statistical analysis of the structures showed that with a confidence greater than 99% the gap
is free of any mass. Fifth, the gap was also seen in 3D maps of yeast ribosome-channel
complexes in intact, not detergent-solubilized membranes (our unpublished observation),
excluding the possibility that it is an artifact introduced by using ribosome-channel complexes
in detergent solution. Preliminary data indicate that the gap is also visible in ribosome-
channel complexes of intact salt-washed mammalian microsomes, providing further evidence
that the ribosome-membrane junction does not change in the presence of a nascent chain.

At the resolution of the 3D maps presented here, the environment in the gap is difficult to
evaluate. It is possible that some low density material is present. Weakly ordered and flexible
segments of either proteins or RNA could reach into the gap. Also, individual α-helices or
amino acid side chains would not be detectable at a resolution of 25-29Å. However, based on
the facts presented above, it seems likely that a gap exists through which nascent chains or
small molecules could pass.
The existence of a gap does not necessarily contradict previous data suggesting that the
ribosome-membrane junction is sealed towards the cytosol. It has been shown

82
Discussion

that polypeptides passing into the lumen of the ER are protected against externally added
proteinase K. Indeed, the gap would be too narrow to allow fully folded proteinase K to enter
it. Likewise, electrophysiological experiments have revealed that in the presence of nascent
chains the flow of ions through the membrane is prevented (Simon and Blobel, 1991).
Assuming that the block occurs within the membrane, possibly mediated by the nascent chain
itself, the gap should not jeopardize the identity of the ER lumen. It is more difficult, however,
to bring our data in agreement with data derived from fluorescence quenching experiments
that suggest the existence of a seal for ions. For example, fluorescent probes in short
preprolactin chains of 56 or 64 amino acids on membrane-bound ribosomes could not be
quenched by iodide ions added to the cytosolic compartment (Crowley et al., 1994). Yet,
these data are in apparent contradiction with the fact that these chains can be trimmed to
about 30 residues by externally added proteinase K (Jungnickel and Rapoport, 1995). So far,
we cannot explain the discrepancy. It seems worth considering that based on the
experimental design it might be much easier to detect the passage of an extended nascent
chain through the gap than the passage of iodide ions, which are supposed to collisionally
quench a fluorescent dye.
The efficiency of collisional quenching depends on the concentration of the quenching agent
and its free diffusion (Hamman et al., 1997). For successful collisional quenching of
fluorescent groups in nascent chains, most likely free diffusion of iodide ions is needed in all 3
dimensions around the fluorescent probe. In addition, the concentration of iodide ions in the
immediate vicinity of the probe must approach the concentration in the bulk solution.
Assuming that hydrated iodide ions have a diameter of about 9Å (Hamman et al., 1998) it is
difficult to envision how these requirements can be fulfilled in a gap that is about 20Å wide.
Furthermore, the negatively charged iodide ions would probably experience some repulsion if
ribosomal RNA is indeed involved in forming the ribosome-membrane junction.
In contrast, for a nascent polypeptide the constrains for entering the gap might be less critical.
A hydrated extended polypeptide chain presumably has a diameter of 10-12Å (Hamman et
al., 1997) suggesting that there might be some steric barriers to entry. However, the nascent
chain might extend toward the cytosol if with ongoing elongation the space between the
ribosomal tunnel exit and the translocon becomes insufficient to accommodate the
polypeptide. Certainly this could be the case for a

83
Discussion

nascent chain with a signal sequence that is too short to insert properly into the translocation
channel. Since this nascent chain cannot induce gating of the channel, the resistance to enter
the plane of the membrane might be greater than the steric hindrance to enter the gap and
subsequently the cytosol. Upon further elongation the nascent chain would reprobe the
translocon, the signal sequence could insert into the membrane and would be recognized by
Sec61p and as a result open the channel for translocation of the polypeptide into the lumen of
the ER.
Once the channel is open, it is probably more favorable for the nascent chain to enter it
instead of looping out into the gap. This could also explain why the energy provided by
protein translation is sufficient to allow directional translocation in the presence of a gap
between ribosome and channel.
When a cytosolic domain of an integral membrane protein is synthesized, its translocation
across the membrane is hindered because the membrane anchor functions as a stop-transfer
sequence. In this case, the gap would permit the exit of the polypeptide into the cytosol
without removal of the ribosome from the translocation site. A similar model would apply to
translocational pausing during synthesis of certain secretory proteins, such as apolipoprotein
B (Hegde and Lingappa, 1996). Here, a non-hydrophobic “pause-transfer” sequence stops
polypeptide movement through the channel and for a short period the newly translated
polypeptide chain gains access to the cytosol, presumably through the gap in the ribosome-
membrane junction.
The gap between ribosome and channel could also be used by membrane-bound ribosomes
translating cytosolic proteins. Since resistance against transfer of these nascent chains
across the membrane is very high, the newly translated cytosolic proteins would probably exit
the ribosome-membrane junction through the gap.

84
Discussion

4.3.2. Shape and Size of the Translocation Channel

Surprisingly, in both ribosome-channel complexes derived from native microsomes or from


vesicles containing purified Sec61p complex we did not detect any significant changes in the
pore size of the translocation channel in the presence or absence of a nascent chain. Our
biochemical data show that the nascent chains are stably bound to the ribosome-channel
complexes. However, no nascent chain or ribosome-associated tRNA was consistently
detected in the 3D structures. This is not surprising since at a resolution of 25-29Å a single
polypeptide chain, that may even be flexible, would not add significant electron-dense mass.
Detection of a ribosome-bound tRNA may be equally difficult in a structure derived from
single particle analysis since the tRNAs in the sample are presumably found at 3 different
sites (Agrawal et al., 1999).

Based on previous biochemical studies it has been suggested that the translocation channel
can exist in two different conformations; as a large pore with an estimated size of 40-60Å
(Hamman et al., 1997) and as a smaller, presumably inactive pore with a size of about 9-15Å
(Hamman et al., 1998).
The largest pore observed in our study was found in channels derived from native
membranes and had a size of about 25x50Å. It may correspond to the large pore described
earlier after insertion of the nascent chain. However, we have not observed the smaller pore
in channels derived from native microsomes, even in the absence of a nascent chain. A
possible explanation is that the structure derived from PKRM without a nascent chain may not
correspond to the true closed state of the translocation channel, maybe due to detergent
solubilization of the membrane during sample preparation.
It is conceivable that the structures derived from purified Sec61p may represent the ground
state, since there the channels have a rather small pore of 15-25Å. However, the size of
these channels did not change in the presence of a nascent chain, maybe suggesting that the
protein that is forming the additional lumenal domain in structures derived from native
membranes is necessary to fully open the translocation channel. This additional protein may
enhance the efficiency of translocation, although it has been shown that translocation can be
reproduced with purified Sec61p complexes in reconstituted proteoliposomes.

85
Discussion

Another interesting difference between native channels and those derived from purified
Sec61p is the shape of the cross-section. Purified Sec61p channels show a cup-like cross-
section with a wider opening towards the ribosome and a somewhat constricted opening
towards the ER lumen. A very similar shape has been described for the Sec61p channel
purified from S. cerevisiae (Beckmann et al., 1997). The narrow lumenal opening might
minimize the transfer of small molecules through the membrane, whereas the wider opening
could collect the growing nascent chain and allow the signal sequence to be in an optimal
position for presentation to the Sec61p complex.
In contrast to the channel derived from purified Sec61p, the native channel shows a much
wider pore that may be more suitable for ongoing translocation. Again, recruitment of the
additional protein into the channel might be necessary to convert it from one state into the
other.
Identification of this additional protein and reconstitution experiments with the purified factor
might help to clarify its role in the assembly of the larger translocation channel.

4.3.3. A Lumenal Domain Associated with the Native Sec61p Channel

In all 3D structures derived from native microsomes, a prominent lumenal domain was
present. It appeared to be precisely oriented with respect to both ribosome and channel. The
tip of the lumenal domain was positioned under the channel pore, as to contact the nascent
chain upon its entry into the ER lumen. We consider it unlikely that this domain is formed by a
soluble protein resident in the ER lumen, since it remains bound to the ribosome-channel
complex even under high salt conditions in the presence of detergent. In addition, as
suggested by a comparison of native and purified channel, the lumenal domain may be part
of an integral membrane protein that is intercalated into the Sec61p ring.
So far, the identity of the protein is unknown. However, we have identified candidate proteins.
Only two membrane protein complexes other than the Sec61p complex are found in the
native ribosome-channel complexes used for single particle analysis. The first candidate is
the oligosaccharyl transferase complex (OST; Kelleher et al., 1992). Of its components we
have found ribophorin I and II and the smaller protein P48 in

86
Discussion

the native ribosome-channel complexes. Since OST transfers carbohydrate chains onto
consensus sites of the nascent chains immediately after its transfer through the membrane, it
is most likely found in close vicinity of the translocation channel. Nevertheless, ribophorin I
and II together with P48 are very likely too large to form the lumenal domain. As a second
complex of membrane proteins we identified the so-called TRAP complex (for translocon-
associated protein; Wiedmann et al., 1987). All four subunits of the TRAP complex have been
shown to be present in ribosome-channel complexes derived from native microsomes. Both
TRAPα and TRAPβ contain large lumenal domains (Görlich et al., 1990). This together with
the overall size of the complex makes TRAP a good candidate for the additional protein
present in translocation channels derived from native membranes. So far, the function of the
TRAP complex is unknown. However, using a photocrosslinking approach it has been shown
that TRAPα is located in close proximity to the nascent chain during its transport across the
membrane (Wiedmann et al., 1987; Krieg et al., 1989; Wiedmann et al., 1989). Although the
TRAP complex is not required for translocation of the secretory protein preprolactin (Görlich
and Rapoport, 1993) and is not essential for translocation of all substrates tested so far
(Migliaccio et al., 1992; Görlich and Rapoport, 1993), it might enhance the performance of the
translocation channel. Since both the TRAP complex and the OST complex have been
isolated, experiments using proteoliposomes containing purified membrane proteins might be
helpful in identifying the partner of Sec61p in translocation of proteins across the ER
membrane.

The data presented in this thesis project have shed initial light on the mechanism of ribosome
binding to the membrane of the endoplasmic reticulum. We have shown that SRP-mediated
targeting comprises more than just guiding the signal sequence-bearing RNCs to the ER
membrane. We have also presented a first structural analysis of the ribosome-translocation
channel complex in the presence of a translocating nascent chain. Based on our data, some
exciting questions are raised. How does a ribosome actively displace another ribosome from
a membrane binding site? What is the role of SRP receptor in translocation channel
assembly? Which other proteins are

87
Discussion

part of the completely assembled channel and what are the dynamics of translocon assembly
and disassembly? How can a translocon “remember” to remain available until the integration
of multi-spanning membrane proteins is completed?
Some of these questions will be answered when a high resolution map of ribosome-
translocation channel complexes is available. A 3D map of ribosomes bound to intact, not
detergent solubilized membranes will also provide more information. Clearly, a more detailed
knowledge of communication between individual components of the translocon will be needed
to better understand this complex system.

88
References

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97
Appendix

6. APPENDIX

ABBREVIATIONS

CAM calmidazolium chloride


CTABr cetyltrimethylammonium bromide
CTF contrast transfer function
DMSO dimethyl sulfoxide
DTT dithiothreitol
ECL enhanced chemiluminescence
EGTA ethylene glycol-bis (β amino ethyl ether)
ER endoplasmic reticulum
eq equivalent (see Walter and Blobel, 1983b)
GDP guanosine 5'-diphosphate
GTP guanosine 5'-triphosphate
Hepes N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
kDa kilodalton
KRM salt washed RM
lep leader peptidase
mRNA messenger-RNA
NAC nascent polypeptide-associated complex
NEM N-ethylmaleimide
PKRM puromycin/high salt treated RM
PMSF phenylmethylsulfonyl fluoride
ppαF preproalphafactor
ppl preprolactin
RM rough microsomes
RNA ribonucleic acid
RNC ribosome-nascent chain complex
rpm revolutions per minute
SDS sodium dodecylsulfate
SDS-PAGE SDS ployacrylamide gel electrophoresis
SRP signal recognition particle
SRP54 54kDa subunit of SRP
SR SRP receptor
TCA trichloroacidic acid
TFP trifluoperazine
TRAM translocating chain associating membrane protein
TRAP translocon associated protein
tRNA transfer RNA
UV ultraviolet light
3D three dimensional

98
Appendix

ERKLÄRUNG

Ich erkläre, daß ich die vorliegende Arbeit selbständig und nur unter Verwendung der angegebenen
Hilfsmittel angefertigt habe.

Andrea Neuhof Boston, den 1.Mai 2000

99
Appendix

LEBENSLAUF

Andrea Neuhof
geboren am 7. Oktober 1971 in Nauen

1990 Erwerb der allgemeinen Hochschulreife (Abitur) an der Erweiterten


Oberschule"G. Dimitroff" in Nauen

1990-1995 Studium der Biochemie an der Humboldt-Universität Berlin

1995 Hochschulabschluß als Diplom-Biochemikerin


Diplomarbeit in der Arbeitsgruppe von Dr. Tom Rapoport am Max-
Delbrück-Centrum für Molekulare Medizin und an der Harvard Medical
School, Boston, zum Thema: "Etablierung eines Assays zum Nachweis von
Phospholipid-Translokasen in der Membran des Endoplasmatischen
Retikulums "

seit 10/1995 Anfertigung der vorliegenden Doktorarbeit in der Arbeitsgruppe von


Dr. Tom Rapoport im Department of Cell Biology,
Harvard Medical School, Boston

100
Appendix

VERÖFFENTLICHUNGEN

Neuhof, A., Rolls, M.M., Jungnickel, B., Kalies, K.U., Rapoport, T.A. (1998) Binding of signal
recognition particle gives ribosome/nascent chain complexes a competitve advantage in endoplasmic
reticulum membrane interaction. Mol. Biol. Cell 9:103-115.

Morgan, D.G., Menetret, J.-F., Radermacher, M., Neuhof, A., Akey, I.V., Rapoport, T.A.,
Akey, C.W. (2000) Comparison of the yeast and rabbit 80S ribosome reveals topology of the
nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments.
J. Mol. Biol. in press.

101
Appendix

DANKSAGUNG

Mein Dank gilt allen ehemaligen und derzeitigen Mitarbeitern des Rapoport-Labors für viele
angenehme und lehrreiche Stunden.

Bei Brigitte, Angelika, Donna, Lara, Linda, Renée und Carol möchte ich mich für die
freundliche Hilfe im Laboralltag bedanken.

Das Strukturprojekt ist eine Kollaboration mit dem Labor von Dr. Chris Akey. Es wäre nicht
durchführbar gewesen ohne Jean-François Ménétret, David Morgan und Chris Akey.

Ein besonderes Dankeschön geht an die unermüdlichen Korrekturleser dieser Arbeit: Kathrin,
Lars, Benjamin, Bill, Melissa, Will und Caroline.

Bei Tom Rapoport bedanke ich mich für seine Unterstützung während meiner Zeit als
Doktorandin und dafür, daß er mir die Gelegenheit gegeben hat, meinen Horizont zu
erweitern.

Vielen Dank an alle, die mich während meiner Jahre in Boston durch Höhen und Tiefen
begleitet haben: Benjamin, Kathrin, Lars, Peter, Mike, Pascal und Berit.

102
Appendix

EIGENANTEIL AN DER VORGELEGTEN ARBEIT

Teil 1: Calmodulin interacts with signal sequences


und
Teil 2: Regulation of ribosome-binding to the ER membrane

Alle gezeigten Experimente wurden von mir durchgeführt.

Teil 3: Structural analysis of ribosome binding to the ER membrane

Dieses Projekt wurde in Zusammenarbeit mit dem Labor von Christopher W. Akey am Boston
University Medical Center durchgeführt.
Ich hatte folgenden Anteil an dem Projekt:
-Reinigung des Sec61p-Komplexes und des SRP-Rezeptors aus rauhen Mikrosomen
-Etablierung und Optimierung der Methodik zur Isolierung von Komplexen aus
Ribosomen/naszierenden Ketten/Translokationskanal, die erstmals die
elektronenmikroskopische Analyse von translozierenden Ribosomen/Sec61p-Komplexen
ermöglichte.
-Präparation aller Proben für die Elektronenmikroskopie.
-Biochemische Analyse der isolierten Ribosomen/Sec61p-Komplexe.
-Die Elektronenmikroskopie und Ermittlung der dreidimensionalen Struktur des
Ribosomen/Sec61p-Komplexes wurden von Dr. Jean-François Ménétret, Dr. David G.
Morgan and Dr. Chris W. Akey durchgeführt.

103